Temporal seed promoters for expressing genes in plants

ABSTRACT

The present invention relates to the field of plant genetic engineering. More specifically, the present invention relates to seed specific gene expression during a defined period of embryogenesis. The present invention provides promoters capable of transcribing heterologous nucleic acid sequences in seeds, and methods of modifying, producing, and using the same.

This application is a divisional of U.S. application No. 11/625,709,filed Jan. 22, 2007, now U.S. Pat. No. 7,615,680 the entire disclosureof which is incorporated herein by reference; which application is acontinuation application of U.S. application Ser. No. 10/429,555 filedMay 5, 2003, issued as U.S. Pat No. 7,179,959 on Feb. 20, 2007; whichclaims the benefit of the filing date of the provisional ApplicationU.S. Ser. No. 60/377,247, filed May 3, 2002.

The present invention relates to the field of plant genetic engineering.More specifically, the present invention relates to seed specific geneexpression with certain temporal profiles during embryogenesis. Theinvention provides promoters capable of transcribing heterologousnucleic acid sequences in seeds, and methods of modifying, producing,and using the same.

Seeds provide and important source of dietary protein for humans andlivestock. However, the nutritional content of seeds is ofteninadequate. For example, many seed proteins are deficient in one or moreessential amino acids. This deficiency may be overcome by geneticallymodifying the native or non-native proteins to have a more nutritionallycomplete composition of amino acids (or some other desirable feature)and to overexpress the modified proteins in the transgenic plants.Alternatively, one or more genes could be introduced into a crop plantto manipulate the metabolic pathways and modify the free amino acidcontent. These approaches are useful in producing crops exhibitingimproved agronomic (e.g., yield), nutritional, and pharmaceuticalproperties.

Introduction of a gene can cause deleterious effects on plant growth anddevelopment. Under such circumstances, the expression of the gene mayneed to be limited to the desired target tissue. For example, it mightbe necessary to express an amino acid deregulation gene in aseed-specific or seed-enhanced fashion to avoid an undesired phenotypethat may affect yield or other agronomic traits. In other cases,expression of the transgene needs to be further limited within a definedperiod of time during the growth and development (temporal profile) ofthe specific target tissue. For example, it might be necessary toexpress a fatty acid metabolic gene during the time when oilbiosynthesis is most active in seed.

The promoter portion of a gene plays a central role in controlling geneexpression. Along the promoter region, the transcription machinery isassembled and transcription is initiated. This early step is often a keyregulatory step relative to subsequent stages of gene expression.Transcription initiation at the promoter may be regulated in severalways. For example, a promoter may be induced by the presence of aparticular compound, express a gene only in a specific tissue, orconstitutively express a coding sequence. Thus, transcription of acoding sequence may be modified by operably linking the coding sequenceto promoters with different regulatory characteristics, the availabilityof which is a problem addressed by this disclosure.

SUMMARY OF THE INVENTION

The present invention includes and provides a substantially purifiednucleic acid molecule encoding a nucleic acid sequence having at least75% identity to SEQ ID NO: 1 or its complement.

The present invention includes and provides a plant comprising anintroduced nucleic acid molecule that comprises a promoter comprising anucleic acid sequence having at least 75% identity to SEQ ID NO: 1 orits complement.

The present invention includes and provides a plant comprising anintroduced nucleic acid molecule that comprises a promoter comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs:1-4 and complements thereof.

The present invention includes and provides a method of producing atransformed plant comprising: providing a nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-4 and complements thereof,operably linked to a structural nucleic acid sequence; and, transforminga plant with said nucleic acid molecule.

The present invention includes and provides a method of expressing astructural nucleic acid molecule in a seed comprising: growing a plantcontaining an introduced nucleic acid molecule that comprises a promotercomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1-4 and complements thereof, operably linked to saidstructural nucleic acid molecule, wherein said plant produces said seedand said structural nucleic acid molecule is transcribed in said seed;and, isolating said seed.

The present invention includes and provides a method of obtaining a seedenhanced in a product of a structural nucleic acid molecule comprising:growing a plant containing an introduced nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-4 and complements thereof,operably linked to said structural nucleic acid molecule, wherein saidtransformed plant produces said seed and said structural nucleic acidmolecule is transcribed in said seed; and, isolating said seed from saidplant.

The present invention includes and provides a method of obtaining mealenhanced in a product of a structural nucleic acid molecule comprising:growing a plant containing an introduced nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-4 and complements thereof,operably linked to said structural nucleic acid molecule, wherein saidplant produces a seed and said structural nucleic acid molecule istranscribed in said seed; and, preparing said meal comprising said plantor part thereof.

The present invention includes and provides a method of obtainingfeedstock enhanced in a product of a structural nucleic acid moleculecomprising: growing a plant containing an introduced nucleic acidmolecule that comprises a promoter comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-4 and complementsthereof, operably linked to said structural nucleic acid molecule,wherein said plant produces a seed and said structural nucleic acidmolecule is transcribed in said seed; and, preparing said feedstockcomprising said plant or part thereof.

The present invention includes and provides a method of obtaining oilenhanced in a product of a structural nucleic acid molecule comprising:growing a plant containing an introduced nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence that hybridizesunder stringent conditions with a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-4 and complements thereof,operably linked to said structural nucleic acid molecule, wherein saidplant produces a seed and said structural nucleic acid molecule istranscribed in said seed; and, isolating said oil.

The present invention includes and provides a cell containing a vectorcomprising a nucleic acid sequence selected from the group consisting ofSEQ ID NOs: 1-4 and complements thereof.

The present invention includes and provides oil produced from one ormore seeds of a plant containing an introduced nucleic acid moleculethat comprises a promoter comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-4 and complements thereof.

The present invention includes and provides oil produced from one ormore seeds of a plant containing an introduced nucleic acid moleculethat comprises a promoter comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-4 and complements thereof,operably linked to a structural nucleic acid sequence, wherein thepromoter is heterologous with respect to the structural nucleic acidsequence.

The present invention includes and provides a seed generated by a plantcontaining an introduced nucleic acid molecule that comprises: apromoter comprising a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs: 1-4 and complements thereof.

The present invention includes and provides feedstock comprising a plantor part thereof containing an introduced nucleic acid molecule thatcomprises a promoter comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 1-4 and complements thereof.

The present invention includes and provides meal comprising plantmaterial from a plant containing an introduced nucleic acid moleculethat comprises a promoter comprising a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 1-4 and complements thereof.

The present invention includes and provides a container of seeds,wherein at least 25% of said seeds comprises a promoter comprising anucleic acid sequence selected from the group consisting of SEQ ID NOs:1-4 and complements thereof, operably linked to a structural nucleicacid sequence, wherein said promoter is heterologous with respect to thestructural nucleic acid sequence.

The present invention includes and provides a method for expressing astructural nucleic acid sequence in a plant, comprising: transformingsaid plant with a nucleic acid molecule comprising a promoter operablylinked to said structural nucleic acid sequence, wherein said promotercomprises a sequence selected from the group consisting of SEQ ID NOs:1-4 and complements thereof, and wherein said promoter and saidstructural nucleic acid sequence are heterologous with respect to eachother, and; growing said plant.

The present invention includes and provides a method of accumulatingfree amino acids in a the seed of a plant, comprising: transforming saidplant with a nucleic acid molecule comprising a promoter operably linkedto a structural nucleic acid sequence encoding an amino acidbiosynthesis gene, wherein said promoter comprises a sequence selectedfrom the group consisting of SEQ ID NOs: 1-4 and complements thereof,and wherein said promoter and said structural nucleic acid sequence areheterologous with respect to each other, and; growing said plant.

The present invention includes and provides a method for regulating thegermination of a soybean seed, comprising transforming a plant with anucleic acid molecule comprising a promoter operably linked to astructural nucleic acid sequence encoding a gibberellin biosyntheticpolypeptide, wherein said promoter comprises a sequence selected fromthe group consisting of SEQ ID NOs: 1-4 and complements thereof, andwherein said promoter and said structural nucleic acid sequence areheterologous with respect to each other; growing said plant; harvestingseed from said plant; and, planting said seed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of pMON8677.

FIG. 2 is a schematic representation of pMON49801.

FIG. 3 is a schematic representation of pMON42316.

FIG. 4 is a schematic representation of pMON42320.

FIG. 5 is a schematic representation of pMON42302.

FIG. 6 is a schematic representation of pMON69650.

FIG. 7 is a schematic representation of pMON69651.

FIG. 8 is a schematic representation of pMON64210.

FIG. 9 is a schematic representation of pMON64213.

FIG. 10 is a schematic representation of pMON66893.

FIG. 11 is a schematic representation of pMON66894.

FIG. 12 is a schematic representation of pMON63662.

FIG. 13 is a schematic representation of pMON63682.

BRIEF DESCRIPTION OF THE SEQUENCES

-   SEQ ID NO: 1 represents a sle2 gene promoter.-   SEQ ID NO: 2 represents a lea9 promoter.-   SEQ ID NO: 3 represents an AtPer1 promoter.-   SEQ ID NO: 4 represents a lectin promoter.-   SEQ ID NOs: 5 to 25 represent primer sequences.

DEFINITIONS

The following definitions are provided as an aid to understanding thedetailed description of the present invention.

The phrases “coding sequence,” “structural sequence,” and “structuralnucleic acid sequence” refer to a physical structure comprising anorderly arrangement of nucleotides. The nucleotides are arranged in aseries of triplets that each form a codon. Each codon encodes a specificamino acid. Thus, the coding sequence, structural sequence, andstructural nucleic acid sequence encode a series of amino acids forminga protein, polypeptide, or peptide sequence. The coding sequence,structural sequence, and structural nucleic acid sequence may becontained within a larger nucleic acid molecule, vector, or the like. Inaddition, the orderly arrangement of nucleotides in these sequences maybe depicted in the form of a sequence listing, figure, table, electronicmedium, or the like.

The phrases “DNA sequence,” “nucleic acid sequence,” and “nucleic acidmolecule” refer to a physical structure comprising an orderlyarrangement of nucleotides. The DNA sequence or nucleotide sequence maybe contained within a larger nucleotide molecule, vector, or the like.In addition, the orderly arrangement of nucleic acids in these sequencesmay be depicted in the form of a sequence listing, figure, table,electronic medium, or the like.

The term “expression” refers to the transcription of a gene to producethe corresponding mRNA and translation of this mRNA to produce thecorresponding gene product (i.e., a peptide, polypeptide, or protein).

The phrase “expression of antisense RNA” refers to the transcription ofa DNA to produce a first RNA molecule capable of hybridizing to a secondRNA molecule.

The term “homology” refers to the level of similarity between two ormore nucleic acid or amino acid sequences in terms of percent ofpositional identity (i.e., sequence similarity or identity). Homologyalso refers to the concept of similar functional properties amongdifferent nucleic acids or proteins.

The term “heterologous” refers to the relationship between two or morenucleic acid or protein sequences that are derived from differentsources. For example, a promoter is heterologous with respect to acoding sequence if such a combination is not normally found in nature.In addition, a particular sequence may be “heterologous” with respect toa cell or organism into which it is inserted (i.e., does not naturallyoccur in that particular cell or organism).

The term “hybridization” refers to the ability of a first strand ofnucleic acid to join with a second strand via hydrogen bond base pairingwhen the two nucleic acid strands have sufficient sequence identity.Hybridization occurs when the two nucleic acid molecules anneal to oneanother under appropriate conditions.

The phrase “operably linked” refers to the functional spatialarrangement of two or more nucleic acid regions or nucleic acidsequences. For example, a promoter region may be positioned relative toa nucleic acid sequence such that transcription of a nucleic acidsequence is directed by the promoter region. Thus, a promoter region is“operably linked” to the nucleic acid sequence.

The term or phrase “promoter” or “promoter region” refers to a nucleicacid sequence, usually found upstream (5′) to a coding sequence, that iscapable of directing transcription of a nucleic acid sequence into mRNA.The promoter or promoter region typically provides a recognition sitefor RNA polymerase and the other factors necessary for proper initiationof transcription. As contemplated herein, a promoter or promoter regionincludes variations of promoters derived by inserting or deletingregulatory regions, subjecting the promoter to random or site-directedmutagenesis, etc. The activity or strength of a promoter may be measuredin terms of the amounts of RNA it produces, or the amount of proteinaccumulation in a cell or tissue, relative to a promoter whosetranscriptional activity has been previously assessed.

The phrase “5′ UTR” refers to the transcribed, but untranslated regionof DNA upstream, or 5′, of the coding region of a gene. A promoter isoften used in combination with its native 5′ UTR in practice. In othercases, a promoter is used in combination with a heterologous 5′ UTR toachieve optimal expression.

The phrase “3′ UTR” refers to the untranslated region of DNA downstream,or 3′, of the coding region of a gene. The 3′ UTR contains signals fortranscription termination and RNA polyadenylation.

The phrase “recombinant vector” refers to any agent such as a plasmid,cosmid, virus, autonomously replicating sequence, phage, or linearsingle-stranded, circular single-stranded, linear double-stranded, orcircular double-stranded DNA or RNA nucleotide sequence. The recombinantvector may be derived from any source and is capable of genomicintegration or autonomous replication.

The phrase “Regulatory sequence” refers to a nucleotide sequence locatedupstream (5′), within, or downstream (3′) to a coding sequence.Transcription and expression of the coding sequence is typicallyimpacted by the presence or absence of the regulatory sequence.

The phrase “substantially homologous” refers to two sequences which areat least about 90% identical in sequence, as measured by the BestFitprogram described herein (Version 10; Genetics Computer Group, Inc.,University of Wisconsin Biotechnology Center, Madison, Wis.), usingdefault parameters.

The term “transformation” refers to the introduction of nucleic acidinto a recipient host. The term “host” refers to bacteria cells, fungi,animals or animal cells, plants or seeds, or any plant parts or tissuesincluding plant cells, protoplasts, calli, roots, tubers, seeds, stems,leaves, seedlings, embryos, and pollen.

As used herein, the phrase “transgenic plant” refers to a plant havingan introduced nucleic acid stably integrated into a genome of the plant,for example, the nuclear or plastid genomes.

As used herein, the phrase “substantially purified” refers to a moleculeseparated from substantially all other molecules normally associatedwith it in its native state. More preferably a substantially purifiedmolecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, preferably75% free, more preferably 90% free, and most preferably 95% free fromthe other molecules (exclusive of solvent) present in the naturalmixture. The term “substantially purified” is not intended to encompassmolecules present in their native state.

In a preferred aspect, a similar genetic background is a backgroundwhere the organisms being compared share 50% or greater of their nucleargenetic material. In a more preferred aspect a similar geneticbackground is a background where the organisms being compared share 75%or greater, even more preferably 90% or greater of their nuclear geneticmaterial. In another even more preferable aspect, a similar geneticbackground is a background where the organisms being compared areplants, and the plants are isogenic except for any genetic materialoriginally introduced using plant transformation techniques.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides promoters capable of transcribing aheterologous structural nucleic acid sequence in a seed, and methods ofmodifying, producing, and using the same. The invention also providescompositions, transformed host cells and plants containing seed specificpromoters, and methods for preparing and using the same.

Nucleic Acid Molecules

The present invention provides nucleic acid molecules comprising asequence selected from the group consisting of SEQ ID NOs: 1-4 andcomplements thereof. SEQ ID NO: 1 represents a sle2 gene promoter. SEQID NO: 2 represents a lea9 promoter. SEQ ID NO: 3 represents an AtPer1promoter. SEQ ID NO: 4 represents a lectin promoter.

Nucleic acid hybridization is a technique well known to those of skillin the art of DNA manipulation. The hybridization property of a givenpair of nucleic acids is an indication of their similarity or identity.

Low stringency conditions may be used to select nucleic acid sequenceswith lower sequence identities to a target nucleic acid sequence. Onemay wish to employ conditions such as about 0.15 M to about 0.9 M sodiumchloride, at temperatures ranging from about 20° C. to about 55° C.

High stringency conditions may be used to select for nucleic acidsequences with higher degrees of identity to the disclosed nucleic acidsequences (Sambrook et al., 1989).

The high stringency conditions typically involve nucleic acidhybridization in about 2× to about 10×SSC (diluted from a 20×SSC stocksolution containing 3 M sodium chloride and 0.3 M sodium citrate, pH 7.0in distilled water), about 2.5× to about 5×Denhardt's solution (dilutedfrom a 50× stock solution containing 1% (w/v) bovine serum albumin, 1%(w/v) ficoll, and 1% (w/v) polyvinylpyrrolidone in distilled water),about 10 mg/mL to about 100 mg/mL fish sperm DNA, and about 0.02% (w/v)to about 0.1% (w/v) SDS, with an incubation at about 50° C. to about 70°C. for several hours to overnight. The high stringency conditions arepreferably provided by 6×SSC, 5×Denhardt's solution, 100 mg/mL fishsperm DNA, and 0.1% (w/v) SDS, with an incubation at 55° C. for severalhours.

The hybridization is generally followed by several wash steps. The washcompositions generally comprise 0.5× to about 10×SSC, and 0.01% (w/v) toabout 0.5% (w/v) SDS with a 15 minute incubation at about 20° C. toabout 70° C. Preferably, the nucleic acid segments remain hybridizedafter washing at least one time in 0.1×SSC at 65° C.

The nucleic acid molecules of the present invention preferablyhybridize, under high stringency conditions, with a nucleic acidmolecule having the sequence selected from the group consisting of SEQID NOs: 1-4 and complements thereof. In a preferred embodiment, anucleic acid molecule of the present invention hybridizes, under highstringency conditions, with a nucleic acid molecule comprising SEQ IDNO: 1. In a preferred embodiment, a nucleic acid molecule of the presentinvention hybridizes, under high stringency conditions, with a nucleicacid molecule comprising SEQ ID NO: 2. In a preferred embodiment, anucleic acid molecule of the present invention hybridizes, under highstringency conditions, with a nucleic acid molecule comprising SEQ IDNO: 3. In a preferred embodiment, a nucleic acid molecule of the presentinvention hybridizes, under high stringency conditions, with a nucleicacid molecule comprising SEQ ID NO: 4.

In a preferred embodiment, a nucleic acid molecule of the presentinvention comprises a nucleic acid sequence that has a sequence identityto SEQ ID NOs: 1, 2, 3, or 4 of greater than about 75, about 80, about85, about 90, about 91, about 92, about 93, about 94, about 95, about96, about 97, about 98, or about 99%.

The percent of sequence identity is preferably determined using the“Best Fit” or “Gap” program of the Sequence Analysis Software Package™(Version 10; Genetics Computer Group, Inc., University of WisconsinBiotechnology Center, Madison, Wis.). “Gap” utilizes the algorithm ofNeedleman and Wunsch (Needleman and Wunsch, 1970) to find the alignmentof two sequences that maximizes the number of matches and minimizes thenumber of gaps. “BestFit” performs an optimal alignment of the bestsegment of similarity between two sequences and inserts gaps to maximizethe number of matches using the local homology algorithm of Smith andWaterman (Smith and Waterman, 1981; Smith et al., 1983). The percentidentity is most preferably determined using the “Best Fit” programusing default parameters.

The present invention also provides nucleic acid molecule fragments thatexhibit a percent identity to any of SEQ ID NO: 1-4 and complementsthereof. In an embodiment, the fragments are between 50 and 600consecutive nucleotides, 50 and 550 consecutive nucleotides, 50 and 500consecutive nucleotides, 50 and 450 consecutive nucleotides, 50 and 400consecutive nucleotides, 50 and 350 consecutive nucleotides, 50 and 300consecutive nucleotides, 50 and 250 consecutive nucleotides, 50 and 200consecutive nucleotides, 50 and 150 consecutive nucleotides, 50 and 100,15 to 100, 15 to 50, or 15 to 25 consecutive nucleotides of a nucleicmolecule of the present invention.

In another embodiment, the fragment comprises at least about 15, 20, 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, or about 650 consecutive nucleotides of a nucleic acidsequence of the present invention.

The present invention contemplates nucleic acid sequences encodingpolypeptides having the enzyme activity of the steroid pathway enzymessqualene epoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α-demethylase, sterol C5 desaturase and sterol methyltransferase II.

Squalene epoxidase (also called squalene monooxygenase) catalyzes theconversion of squalene to squalene epoxide (2,3-oxidosqualene), aprecursor to the initial sterol molecule in phytosterol biosyntheticpathway, cycloartenol. This is the first reported step in the pathwaywhere oxygen is required for activity. The formation of squalene epoxideis also the last common reported step in sterol biosynthesis of animals,fungi and plants. Recently, several homologues of Arabidopsis andBrassica squalene epoxidase genes are reported (Schafer, U. A.; Reed, D.W.; Hunter, D. G.; Yao, K.; Weninger, A. M.; Tsang, E. W.; Reaney, M.J.; MacKenzie, S. L.; and Covello, P. S. (1999) Plant Mol. Biol.,39(4):721-728). The same authors also have PCT application disclosingthe use of antisense technology with squalene epoxidase to elevatesqualene levels in plants (WO 97/34003).

Squalene Epoxidase, also known as squalene monooxygenase is enzymereference number 1.14.99.7, Enzyme Nomenclature, 1992, p. 146.

Several squalene epoxidase enzymes are known to the art. These includeArabidopsis squalene epoxidase protein sequence Accession No. AC004786,Arabidopsis squalene epoxidase Accession No. N64916, and Arabidopsissqualene epoxidase Accession No. T44667. Japanese Patent Application 07194 381 A discloses a DNA encoding a mammalian squalene epoxidase.

An additional aspect of the invention is the recombinant constructs andvectors comprising nucleic acid sequences encoding squalene epoxidase,as well as a method of producing the novel squalene epoxidase,comprising culturing a host cell transformed with the novel constructsor vectors for a time and under conditions conductive to the productionof the squalene epoxidase, and recovering the squalene epoxidaseproduced thereby.

S-adenosyl-L-methionine:sterol C24 methyl transferases (SMT1 and SMT2)catalyze the transfer of a methyl group from a cofactor,S-adenosyl-L-methionine, to the C24 center of the sterol side chain(Bach, T. J. and Benveniste, P. (1997), Prog. Lipid Res., 36:197-226).SMT in higher plant cells are responsible for their capability toproduce a mixture of 24-methyl and 24-ethyl sterols (Schaffer, A.;Bouvier-Navé, Benveniste, P.; Schaller, H.; (2000) Lipids, 35:263-269).Functional characterization of the SMT using a yeast erg6 expressionsystem demonstrated unambiguously that an SMT1 sequence encodes acycloartenol-C24-methyltransferase and a SMT2 sequence encodes a24-methylene lophenol-C24-methyltransferase in a given plant species(Bouvier-Navé, P.; Husselstein, T.; and Benveniste, P. (1998), Eur. J.Biochem., 246:518-529). Several plant genes coding for SMT1 and SMT2have been reported and reviewed (Schaffer, A.; Bouvier-Navé, Benveniste,P.; Schaller, H. (2000) Lipids, 35:263-269). Transgenic plantsexpressing homologues of either SMT1 or SMT2 have been studied(Schaffer, A.; Bouvier-Navé, Benveniste, P.; Schaller, H. (2000) Lipids,35:263-269). The use of these genes to modify plant sterol compositionare also covered by two patent applications (WO 98/45457 and WO00/61771).

Sterol methyl transferase I enzymes known in the art are useful in thepresent invention. Exemplary sequences include the known Arabidopsissterol methyl transferase I protein sequence Accession No. U71400, theknown tobacco sterol methyl transferase I protein sequence Accession No.U81312 and Ricinus communis sterol C methyltransferase, Eur. J.Biochem., 246(2); 518-529 (1997). (Complete cds, Accession No.g2246457).

S-Adenosyl-L-Methionine Sterol C24 Methyltransferase—A nucleic acidsequence encoding an Arabidopsis thaliana S-adenosyl-L-methionine-sterolC24 methyltransferase has been published by Husselstein et al. (1996)FEBS Letters 381:87-92. Δ²⁴-sterol C-methyltransferase is enzyme number2.1.1.41, Enzyme Nomenclature 1992, page 160.

Sterol C4 demethylase catalyses the first of several demethylationreactions, which results in the removal of the two methyl groups at C4.While in animals and fungi the removal of the two C4 methyl groupsoccurs consecutively, in plants it has been reported that there areother steps between the first and second C4 demethylations (Bach, T. J.and Benveniste, P. (1997), Prog. Lipid Res., 36:197-226). The C4demethylation is catalyzed by a complex of microsomal enzymes consistingof a monooxygenase, an NAD⁺-dependent sterol C4 decarboxylase, and anNADPH-dependent 3-ketosteroid reductase.

Sterol C14 demethylase catalyzes demethylation at C14 which removes themethyl group at C14 and creates a double bond at that position. In bothfungi and animals, this is the first step in the sterol synthesispathway. However, in higher plants, the 14α-methyl is removed after oneC4 methyl has disappeared. Thus, while lanosterol is the substrate forC14 demethylase in animal and fungal cells, the plants enzyme usesobtusifoliol as substrate. Sterol C14 demethylation is mediated by acytochrome P-450 complex. The mechanism of 14-methyl removal involvestwo oxidation steps leading to an alcohol, then an aldehyde at C29 and afurther oxidative step involving a deformylation leading to formic acidand the sterol product with a typical 8, 14-diene (Aoyama, Y.; Yoshida,Y.; Sonoda, Y.; and Sato, Y. (1989) J. Biol. Chem., 264:18502-18505).Obtusifoliol C14 α-demethylase from Sorghum bicolor (L) Moench has beencloned using a gene-specific probe generated using PCR primers designedfrom an internal 14 amino acid sequence and was functionally expressedin E. coli (Bak, S.; Kahn, R. A.; Olsen, C. E.; and Halkier, B. A.(1997) The Plant Journal, 11(2):191-201). Also, Saccharomyces cerevisiaeCYP51A1 encoding lanosterol C14 demethylase was functionally expressedin tobacco (Grausem, B.; Chaubet, N.; Gigot, C.; Loper, J. C.; andBenveniste, P. (1995) The Plant Journal, 7(5):761-770).

Sterol C14 demethylase enzymes and sequences are known in the art. Forexample Sorghum bicolor obtusifoliol C14 α-demethylase CYP51 mRNA,described in Plant J., 11(2):191-201 (1997) (complete cds Acession No.U74319).

An additional aspect of the invention is the recombinant constructs andvectors comprising nucleic acid sequences encoding obtusifoliol C14α-demethylase, as well as a method of producing obtusifoliol C14α-demethylase, comprising culturing a host cell transformed with thenovel constructs or vectors for a time and under conditions conductiveto the production of the obtusifoliol C14 α-demethylase, and recoveringthe obtusifoliol C14 α-demethylase produced thereby.

Sterol C5 desaturase catalyzes the insertion of the Δ⁵-double bond thatnormally occurs at the Δ⁷-sterol level, thereby forming a Δ^(5,7)-sterol(Parks et al., Lipids, 30:227-230 (1995)). The reaction has beenreported to involve the stereospecific removal of the 5α and 6α hydrogenatoms, biosynthetically derived from the 4 pro-R and 5 pro-S hydrogensof the (+) and (−) R-mevalonic acid, respectively (Goodwin, T. W. (1979)Annu. Rev. Plant Physiol., 30:369-404). The reaction is obligatorilyaerobic and requires NADPH or NADH. The desaturase has been reported tobe a multienzyme complex present in microsomes. It consists of thedesaturase itself, cytochrome b₅ and a pyridine nucleotide-dependentflavoprotein. The Δ⁵-desaturase is reported to be a mono-oxygenase thatutilizes electrons derived from a reduced pyridine nucleotide viacytochrome_(b) (Taton, M., and Rahier, A. (1996) Arch. Biochem.Biophys., 325:279-288). An Arabidopsis thaliana cDNA encoding a sterolC5 desaturase was cloned by functional complementation of a yeastmutant, erg3 defective in ERG3, the gene encoding the sterol C5desaturase required for ergosterol biosynthesis (Gachotte D.;Husselstein, T.; Bard, M.; Lacroute F.; and Benveniste, P. (1996) ThePlant Journal, 9(3):391-398). Known sterol C5 desaturase enzymes areuseful in the present invention, including Arabidopsis sterol C5desaturase protein sequence Accession No. X90454, and the Arabidopsisthaliana mRNA for sterol C5 desaturase described in Plant J.9(3):391-398 (1996) (complete cds Accession No. g1061037).

The NCBI (National Center for Biotechnology Information) database shows37 sequences for sterol desaturase that are useful in the presentinvention. The following are exemplary of such sequences. From yeast: C5sterol desaturase NP_(—)013157 (Saccharomyces cerevisiae); hypotheticalC5 sterol desaturase-fission T40027 (Schizosaccharomyces pombe); C5sterol desaturase-fission T37759 (Schizosaccharomyces pombe); C5 steroldesaturase JQ1146 (Saccharomyces cerevisiae); C5 sterol desaturaseBAA21457 (Schizosaccharomyces pombe); C5 sterol desaturase CAA22610(Schizosaccharomyces pombe); putative C5 sterol desaturase CAA16898(Schizosaccharomyces pombe); probable C5 sterol desaturase O13666(erg3_schpo); C5 sterol desaturase P50860 (Erg3_canga); C5 steroldesaturase P32353 (erg3_yeast); C5,6 desaturase AAC99343 (Candidaalbicans); C5 sterol desaturase BAA20292 (Saccharomyces cerevisiae); C5sterol desaturase AAB39844 (Saccharomyces cerevisiae); C5 steroldesaturase AAB29844 (Saccharomyces cerevisiae); C5 sterol desaturaseCAA64303 (Saccharomyces cerevisiae); C5 sterol desaturase AAA34595(Saccharomyces cerevisiae); C5 sterol desaturase AAA34594 (Saccharomycescerevisiae). From plants: C5 sterol desaturase S71251 (Arabidopsisthaliana); putative sterol C5 desaturase AAF32466 (Arabidopsisthaliana); sterol C5 desaturase AAF32465 (Arabidopsis thaliana);putatuve sterol des aturase AAF22921 (Arabidopsis thaliana); Δ⁷-sterolC5 desaturase (Arabidopsis thaliana); sterol C5(6) desaturase homologAAD20458 (Nicotiana tabacum); sterol C5 desaturase AAD12944 (Arabidopsisthaliana); sterol C5,6 desaturase AAD04034 (Nicotiana tabacum); sterolC5 desaturase CAA62079 (Arabidopsis thaliana). From mammals: sterol C5desaturase (Mus musculus) BAA33730; sterol C5 desaturase BAA33729 (Homosapiens); lathosterol oxidase CAB65928 (Leishmania major); lathosteroloxidase (lathosterol 5 desaturase) O88822 (Mus musculus); lathosterol 5desaturase O75845 (Homo sapiens); Δ⁷-sterol C5 desaturase AAF00544 (Homosapiens). Others: fungal sterol C5 desaturase homolog BAA18970 (Homosapiens).

For DNA sequences encoding a sterol C5 desaturase useful in the presentinvention, the NCBI nucleotide search for “sterol desaturase” came upwith 110 sequences. The following are exemplary of such sequences.NC_(—)001139 (Saccharomyces cerevisiae); NC_(—)001145 (Saccharomycescerevisiae); NC_(—)001144 (Saccharomyces cerevisiae); AW700015(Physcomitrella patens); AB004539 (Schizosaccharomyces pombe); andAW596303 (Glycine max); AC012188 (Arabidopsis thaliana).

The combination of introduction of an HMG-CoA reductase gene along witha sterol methyl transferase II gene into a cell serves to reduce steroidpathway intermediate compound accumulation in addition to reducing theaccumulation of 24-methyl sterols such as campesterol.

Known sterol methyl transferase II enzymes are useful in the presentinvention, including Arabidopsis sterol methyl transferase II proteinsequence (complete mRNA cds from FEBS Lett., 381(12):87-92 (1996)Accession No. X89867).

Recombinant constructs encoding any of the forgoing enzymes affectingthe steroid biosynthetic pathway can be incorporated into recombinantvectors comprising the recombinant constructs comprising the isolatedDNA molecules. Such vectors can be bacterial or plant expressionvectors.

In a preferred embodiment, any of the plants or organisms of the presentinvention are transformed with a nucleic acid of the present inventionand a gene encoding a member selected from the group consisting ofsqualene epoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α-demethylase, sterol C5 desaturase, and sterol methyltransferase II. In a preferred embodiment, a plant or organism of thepresent invention is transformed with one or more of SEQ ID NOs: 1-4 anda gene encoding a member selected from the group consisting of squaleneepoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α-demethylase, sterol C5 desaturase, and sterol methyltransferase II. In a further preferred embodiment, a plant or organismof the present invention is transformed with one or more of SEQ ID NOs:1-4, a gene encoding a member selected from the group consisting ofsqualene epoxidase, sterol methyl transferase I, sterol C4 demethylase,obtusifoliol C14 α-demethylase, sterol C5 desaturase, and sterol methyltransferase II, and one or more genes encoding a tocopherol pathwayenzyme as disclosed elsewhere herein. In a further preferred embodiment,a plant or organism of the present invention is transformed with one ormore of SEQ ID NOs: 1-4, two genes encoding a member selected from thegroup consisting of squalene epoxidase, sterol methyl transferase I,sterol C4 demethylase, obtusifoliol C14 α-demethylase, sterol C5desaturase, and sterol methyl transferase II, and two genes encoding atocopherol pathway enzyme as disclosed elsewhere herein. Any of theabove combinations of tocopherol and sterol biosynthesis genes can beintroduced into a plant on one or more constructs or vectors, as isknown in the art and described herein.

Promoters

In one embodiment any of the disclosed nucleic acid molecules may bepromoters. In a preferred embodiment, the promoter is tissue or organspecific, and preferably seed specific. In a particularly preferredembodiment the promoter preferentially expresses associated structuralgenes in the endosperm or embryo. In a preferred embodiment, thepromoter preferentially drives expression of the associated structuralgenes during a defined period of embryogenesis in seed.

In one aspect, a promoter is considered tissue or organ specific if thelevel of an mRNA in that tissue or organ is expressed at a level that isat least 10 fold higher, preferably at least 100 fold higher or at least1,000 fold higher than another tissue or organ. The level of mRNA can bemeasured either at a single time point or at multiple time points and assuch the fold increase can be average fold increase or an extrapolatedvalue derived from experimentally measured values. As it is a comparisonof levels, any method that measures mRNA levels can be used. In apreferred aspect, the tissue or organs compared are a seed or seedtissue with a leaf or leaf tissue. In another preferred aspect, multipletissues or organs are compared. A preferred multiple comparison is aseed or seed tissue compared with two, three, four or more tissues ororgans selected from the group consisting of floral tissue, floral apex,pollen, leaf, embryo, shoot, leaf primordia, shoot apex, root, root tip,vascular tissue and cotyledon. As used herein, examples of plant organsare seed, leaf, root, etc. and example of tissues are leaf primordia,shoot apex, vascular tissue etc. The activity or strength of a promotermay be measured in terms of the amount of mRNA or protein accumulationit specifically produces, relative to the total amount of mRNA orprotein at a single time point or at multiple time points in thespecific tissue during the growth and development. The temporal profileof a promoter may be obtained by compare the relative strength of thepromoter in the tissues collected at multiple time points during thegrowth and development.

Alternatively, the temporal profile of a promoter may be expressedrelative to a well-characterized promoter (for which expression profilewas previously assessed). For example, a promoter of interest may beoperably linked to a reporter sequence (e.g., GUS) and introduced into aspecific cell type. A known promoter may be similarly prepared andintroduced into the same cellular context. Transcriptional activity ofthe promoter of interest is then determined by comparing the timing ofreporter expression, relative to that of the known promoter. Thecellular context is preferably soybean.

Structural Nucleic Acid Sequences

The promoters of the present invention may be operably linked to astructural nucleic acid sequence that is heterologous with respect tothe promoter. The structural nucleic acid sequence may generally be anynucleic acid sequence for which an increased level of transcription isdesired. The structural nucleic acid sequence preferably encodes apolypeptide that is suitable for incorporation into the diet of a humanor an animal or provides some other agriculturally important feature.

Suitable structural nucleic acid sequences include, without limitation,those encoding seed storage proteins, fatty acid pathway enzymes,tocopherol biosynthetic enzymes, amino acid biosynthetic enzymes,steroid pathway enzymes, and starch branching enzymes.

Preferred seed storage proteins include zeins (U.S. Pat. Nos. 4,886,878;4,885,357; 5,215,912; 5,589,616; 5,508,468; 5,939,599; 5,633,436; and5,990,384; Patent Applications WO 90/01869, WO 91/13993, WO 92/14822, WO93/08682, WO 94/20628, WO 97/28247, WO 98/26064, and WO 99/40209), 7Sproteins (U.S. Pat. Nos. 5,003,045 and 5,576,203), Brazil nut protein(U.S. Pat. No. 5,850,024), phenylalanine free proteins (PatentApplication WO 96/17064), albumin (Patent Application WO 97/35023),β-conglycinin (Patent Application WO 00/19839), 11S (U.S. Pat. No.6,107,051), α-hordothionin (U.S. Pat. Nos. 5,885,802 and 5,885,801),arcelin seed storage proteins (U.S. Pat. No. 5,270,200), lectins (U.S.Pat. No. 6,110,891), and glutenin (U.S. Pat. Nos. 5,990,389 and5,914,450).

Preferred fatty acid pathway enzymes include thioesterases (U.S. Pat.Nos. 5,512,482; 5,530,186; 5,945,585; 5,639,790; 5,807,893; 5,955,650;5,955,329; 5,759,829; 5,147,792; 5,304,481; 5,298,421; 5,344,771; and5,760,206), and desaturases (U.S. Pat. Nos. 5,689,050; 5,663,068;5,614,393; 5,856,157; 6,117,677; 6,043,411; 6,194,167; 5,705,391;5,663,068; 5,552,306; 6,075,183; 6,051,754; 5,689,050; 5,789,220;5,057,419; 5,654,402; 5,659,645; 6,100,091; 5,760,206; 6,172,106;5,952,544; 5,866,789; 5,443,974; and 5,093,249). Preferred tocopherolbiosynthetic enzymes include tyrA, slr1736, ATPT2, dxs, dxr, GGPPS,HPPD, GMT, MT1, tMT2, AANT1, slr1737, and an antisense construct forhomogentisic acid dioxygenase (Kridl et al., Seed Sci. Res., 1:209:219(1991); Keegstra, Cell, 56(2):247-53 (1989); Nawrath, et al., Proc.Natl. Acad. Sci. (U.S.A.), 91:12760-12764 (1994); Xia et al., J. Gen.Microbiol., 138:1309-1316 (1992); Cyanobasehttp://www.kazusa.or.jp/cyanobase; Lois et al., Proc. Natl. Acad. Sci.(U.S.A.), 95(5):2105-2110 (1998); Takahashi et al. Proc. Natl. Acad.Sci. (U.S.A.), 95(17):9879-9884 (1998); Norris et al., Plant Physiol.,117:1317-1323 (1998); Bartley and Scolnik, Plant Physiol., 104:1469-1470(1994); Smith et al., Plant J., 11:83-92 (1997); WO 00/32757; WO00/10380; Saint Guily, et al., Plant Physiol., 100(2):1069-1071 (1992);Sato et al., J. DNA Res., 7(1):31-63 (2000)).

Various genes and their encoded proteins that are involved in tocopherolbiosynthesis are listed in the table below.

Gene ID Enzyme name tyrA Prephanate dehydrogenase slr1736 Phytylprenyltransferase from Synechocystis ATPT2 Phytylprenyl transferase fromArabidopsis thaliana DXS 1-Deoxyxylulose-5-phosphate synthase DXR1-Deoxyxylulose-5-phosphate reductoisomerase GGPPS Geranylgeranylpyrophosphate synthase HPPD p-Hydroxyphenylpyruvate dioxygenase AANT1Adenylate transporter slr1737 Tocopherol cyclase IDI Isopentenyldiphosphate isomerase GGH Geranylgeranyl reductase GMT Gamma MethylTransferase

The “Gene IDs” given in the table above identify the gene associatedwith the listed enzyme. Any of the Gene IDs listed in the tableappearing herein in the present disclosure refer to the gene encodingthe enzyme with which the Gene ID is associated in the table.

Preferred amino acid biosynthetic enzymes include anthranilate synthase(U.S. Pat. No. 5,965,727; Patent Applications WO 97/26366, WO 99/11800,and WO 99/49058), tryptophan decarboxylase (Patent Application WO99/06581), threonine decarboxylase (U.S. Pat. Nos. 5,534,421 and5,942,660; Patent Application WO 95/19442), threonine deaminase (PatentApplications WO 99/02656, and WO 98/55601), and aspartate kinase (U.S.Pat. Nos. 5,367,110; 5,858,749; and 6,040,160).

Preferred starch branching enzymes include those set forth in U.S. Pat.Nos. 6,232,122 and 6,147,279; and Patent Application WO 97/22703.

Alternatively, a promoter and structural nucleic acid sequence may bedesigned to down-regulate a specific nucleic acid sequence. This istypically accomplished by linking the promoter to a structural nucleicacid sequence that is oriented in the antisense direction. One ofordinary skill in the art is familiar with such antisense technology.Any nucleic acid sequence may be negatively regulated in this manner.

Targets of such regulation may include polypeptides that have a lowcontent of essential amino acids, yet are expressed at a relatively highlevel in a particular tissue. For example, β-conglycinin and glycininare expressed abundantly in seeds, but are nutritionally deficient withrespect to essential amino acids. This antisense approach may also beused to effectively remove other undesirable proteins, such asantifeedants (e.g., lectins), albumin, and allergens, from plant-derivedfeed or to down-regulate catabolic enzymes involved in degradation ofdesired compounds such as essential amino acids.

Modified Structural Nucleic Acid Sequences

The promoters of the present invention may also be operably linked to amodified structural nucleic acid sequence that is heterologous withrespect to the promoter. The structural nucleic acid sequence may bemodified to provide various desirable features. For example, astructural nucleic acid sequence may be modified to increase the contentof essential amino acids, enhance translation of the amino acidsequence, alter post-translational modifications (e.g., phosphorylationsites), transport a translated product to a compartment inside oroutside of the cell, improve protein stability, insert or delete cellsignaling motifs, increase plant cell or organ size, and the like.

In a preferred embodiment, the structural nucleic acid sequence isenhanced to encode a polypeptide having an increased content of at leastone, and more preferably 2, 3, or 4 of the essential amino acidsselected from the group consisting of histidine, lysine, methionine, andphenylalanine. Non-essential amino acids may also be added, as needed,for structural and nutritive enhancement of the polypeptide. Structuralnucleic acid sequences particularly suited to such enhancements includethose encoding native polypeptides that are expressed at relatively highlevels, have a particularly low content of essential amino acids, orboth. Examples of such are the seed storage proteins, such as glycininand β-conglycinin. Other suitable targets include arcelin, phaseolin,lectin, zeins, and albumin.

Codon Usage in Structural Nucleic Acid Sequences

Due to the degeneracy of the genetic code, different nucleotide codonsmay be used to code for a particular amino acid. A host cell oftendisplays a preferred pattern of codon usage. Structural nucleic acidsequences are preferably constructed to utilize the codon usage patternof the particular host cell. This generally enhances the expression ofthe structural nucleic acid sequence in a transformed host cell. Any ofthe above described nucleic acid and amino acid sequences may bemodified to reflect the preferred codon usage of a host cell or organismin which they are contained. Modification of a structural nucleic acidsequence for optimal codon usage in plants is described in U.S. Pat. No.5,689,052.

Other Modifications of Structural Nucleic Acid Sequences

Additional variations in the structural nucleic acid sequences describedabove may encode proteins having equivalent or superior characteristicswhen compared to the proteins from which they are engineered. Mutationsmay include deletions, insertions, truncations, substitutions, fusions,shuffling of motif sequences, and the like.

Mutations to a structural nucleic acid sequence may be introduced ineither a specific or random manner, both of which are well known tothose of skill in the art of molecular biology. A myriad ofsite-directed mutagenesis techniques exist, typically usingoligonucleotides to introduce mutations at specific locations in astructural nucleic acid sequence. Examples include single strand rescue(Kunkel et al., 1985), unique site elimination (Deng and Nickloff,1992), nick protection (Vandeyar et al., 1988), and PCR (Costa et al.,1996). Random or non-specific mutations may be generated by chemicalagents (for a general review, see Singer and Kusmierek, 1982) such asnitrosoguanidine (Cerda-Olmedo et al., 1968; Guerola et al., 1971) and2-aminopurine (Rogan and Bessman, 1970); or by biological methods suchas passage through mutator strains (Greener et al., 1997). Additionalmethods of making the alterations described above are described byAusubel et al. (1995); Bauer et al. (1985); Craik (1985); Frits Ecksteinet al. (1982); Sambrook et al. (1989); Smith et al. (1981); and Osuna etal. (1994).

The modifications may result in either conservative or non-conservativechanges in the amino acid sequence. Conservative changes are changeswhich do not alter the final amino acid sequence of the protein. In apreferred embodiment, the protein has between about 5 and about 500conservative changes, more preferably between about 10 and about 300conservative changes, even more preferably between about 25 and about150 conservative changes, and most preferably between about 5 and about25 conservative changes or between about 1 and about 5 conservativechanges.

Non-conservative changes include additions, deletions, and substitutionswhich result in an altered amino acid sequence. In a preferredembodiment, the protein has between about 5 and about 500non-conservative amino acid changes, more preferably between about 10and about 300 non-conservative amino acid changes, even more preferablybetween about 25 and about 150 non-conservative amino acid changes, andmost preferably between about 5 and about 25 non-conservative amino acidchanges or between about 1 and about 5 non-conservative changes.

Modifications may be made to the protein sequences described herein andthe nucleic acid sequences which encode them that maintain the desiredproperties of the molecule. The following is a discussion based uponchanging the amino acid sequence of a protein to create an equivalent,or possibly an improved, second-generation molecule. The amino acidchanges may be achieved by changing the codons of the structural nucleicacid sequence, according to the codons given in the table below.

Codon Degeneracy of Amino Acids

One Three Amino acid letter letter Codons Alanine A Ala GCA GCC GCG GCTCysteine C Cys TGC TGT Aspartic acid D Asp GAC GAT Glutamic acid E GluGAA GAG Phenylalanine F Phe TTC TTT Glycine G Gly GGA GGC GGG GGTHistidine H His CAC CAT Isoleucine I Ile ATA ATC ATT Lysine K Lys AAAAAG Leucine L Leu TTA TTG CTA CTC CTG CTT Methionine M Met ATGAsparagine N Asn AAC AAT Proline P Pro CCA CCC CCG CCT Glutamine Q GlnCAA CAG Arginine R Arg AGA AGG CGA CGC CGG CGT Serine S Ser AGC AGT TCATCC TCG TCT Threonine T Thr ACA ACC ACG ACT Valine V Val GTA GTC GTG GTTTryptophan W Trp TGG Tyrosine Y Tyr TAC TAT

Certain amino acids may be substituted for other amino acids in aprotein sequence without appreciable loss of the desired activity. It isthus contemplated that various changes may be made in peptide sequencesor protein sequences, or their corresponding nucleic acid sequenceswithout appreciable loss of the biological activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982). It is accepted thatthe relative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics. These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2);glutamate/glutamine/aspartate/asparagine (−3.5); lysine (−3.9); andarginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, (i.e., stillobtain a biologically functional protein). In making such changes, thesubstitution of amino acids whose hydropathic indices are within ±2 ispreferred, those within ±1 are more preferred, and those within ±0.5 aremost preferred.

It is also understood in the art that the substitution of like aminoacids may be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101 states that the greatest local average hydrophilicity of aprotein, as governed by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. The followinghydrophilicity values have been assigned to amino acids: arginine/lysine(+3.0); aspartate/glutamate (+3.0±1); serine (+0.3);asparagine/glutamine (+0.2); glycine (0); threonine (−0.4); proline(−0.5±1); alanine/histidine (−0.5); cysteine (−1.0); methionine (−1.3);valine (−1.5); leucine/isoleucine (−1.8); tyrosine (−2.3); phenylalanine(−2.5); and tryptophan (−3.4).

It is understood that an amino acid may be substituted by another aminoacid having a similar hydrophilicity score and still result in a proteinwith similar biological activity, (i.e., still obtain a biologicallyfunctional protein). In making such changes, the substitution of aminoacids whose hydropathic indices are within ±2 is preferred, those within±1 are more preferred, and those within ±0.5 are most preferred.

As outlined above, amino acid substitutions are therefore based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions which take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine, andisoleucine. Changes which are not expected to be advantageous may alsobe used if these resulted proteins have improved rumen resistance,increased resistance to proteolytic degradation, or both improved rumenresistance and increased resistance to proteolytic degradation, relativeto the unmodified polypeptide from which they are engineered.Alternatively, changes could be made to improve kinetics of metabolicenzymes.

In a preferred aspect, the protein modified is selected from seedstorage proteins, fatty acid pathway enzymes, tocopherol biosyntheticenzymes, amino acid biosynthetic enzymes and starch branching enzymes.

Recombinant Vectors

Any of the promoters and structural nucleic acid sequences describedabove may be provided in a recombinant vector. A recombinant vectortypically comprises, in a 5′ to 3′ orientation: a promoter to direct thetranscription of a structural nucleic acid sequence and a structuralnucleic acid sequence. Suitable promoters and structural nucleic acidsequences include those described herein. The recombinant vector mayfurther comprise a 3′ transcriptional terminator, a 3′ polyadenylationsignal, other untranslated nucleic acid sequences, transit and targetingnucleic acid sequences, selectable markers, enhancers, and operators, asdesired.

Means for preparing recombinant vectors are well known in the art.Methods for making recombinant vectors particularly suited to planttransformation are described in U.S. Pat. Nos. 4,971,908; 4,940,835;4,769,061; and 4,757,011. These types of vectors have also been reviewed(Rodriguez et al., 1988; Glick et al., 1993).

Typical vectors useful for expression of nucleic acids in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers et al.,1987). Other recombinant vectors useful for plant transformation,including the pCaMVCN transfer control vector, have also been described(Fromm et al., 1985).

In one embodiment, multiple promoters are operably linked in a singleconstruct to any combination of structural genes. In a preferredembodiment, any combination of 1, 2, 3, 4, 5, or 6 or more of nucleicacid molecules comprising SEQ ID NOs: 1-4 can be operatively linked in asingle construct to any combination of structural genes. In anotheraspect of the preferred embodiment, the nucleic acid molecules may bemodified. Such modifications can include, for example, removal oraddition of one or more structural or functional elements.

Additional Promoters in the Recombinant Vector

One or more additional promoters may also be provided in the recombinantvector. These promoters may be operably linked, for example, withoutlimitation, to any of the structural nucleic acid sequences describedabove. Alternatively, the promoters may be operably linked to othernucleic acid sequences, such as those encoding transit peptides,selectable marker proteins, or antisense sequences.

These additional promoters may be selected on the basis of the cell typeinto which the vector will be inserted. Also, promoters which functionin bacteria, yeast, and plants are all well taught in the art. Theadditional promoters may also be selected on the basis of theirregulatory features. Examples of such features include enhancement oftranscriptional activity, inducibility, tissue specificity, anddevelopmental stage-specificity. In plants, promoters that areinducible, of viral or synthetic origin, constitutively active,temporally regulated, and spatially regulated have been described(Poszkowski et al., 1989; Odell et al., 1985; Chau et al., 1989).

Often-used constitutive promoters include the CaMV 35S promoter (Odellet al., 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus(FMV) promoter (Richins et al., 1987), the mannopine synthase (mas)promoter, the nopaline synthase (nos) promoter, and the octopinesynthase (ocs) promoter.

Useful inducible promoters include promoters induced by salicylic acidor polyacrylic acids (PR-1; Williams et al., 1992), induced byapplication of safeners (substituted benzenesulfonamide herbicides;Hershey and Stoner, 1991), heat-shock promoters (Ou-Lee et al., 1986;Ainley et al., 1990), a nitrate-inducible promoter derived from thespinach nitrite reductase structural nucleic acid sequence (Back et al.,1991), hormone-inducible promoters (Yamaguchi-Shinozaki et al., 1990;Kares et al., 1990), and light-inducible promoters associated with thesmall subunit of RuBP carboxylase and LHCP families (Kuhlemeier et al.,1989; Feinbaum et al., 1991; Weisshaar et al., 1991; Lam and Chua, 1990;Castresana et al., 1988; Schulze-Lefert et al., 1989).

Examples of useful tissue or organ specific promoters includeβ-conglycinin, (Doyle et al., 1986; Slighton and Beachy, 1987), andother seed specific promoters (Knutzon et al., 1992; Bustos et al.,1991; Lam and Chua, 1991). Plant functional promoters useful forpreferential expression in seed include those from plant storageproteins and from proteins involved in fatty acid biosynthesis inoilseeds. Examples of such promoters include the 5′ regulatory regionsfrom such structural nucleic acid sequences as napin (Kridl et al.,1991), phaseolin, zein, soybean trypsin inhibitor, ACP, stearoyl-ACPdesaturase, and oleosin. Seed-specific regulation is further discussedin European Patent 0 255 378.

Another exemplary seed specific promoter is a lectin promoter. Thelectin protein in soybean seeds is encoded by a single structuralnucleic acid sequence (Le1) that is only expressed during seeddevelopment. A lectin structural nucleic acid sequence and seed-specificpromoter have been characterized and used to direct seed specificexpression in transgenic tobacco plants (Vodkin et al., 1983; Lindstromet al., 1990).

Particularly preferred additional promoters in the recombinant vectorinclude the nopaline synthase (nos), mannopine synthase (mas), andoctopine synthase (ocs) promoters, which are carried on tumor-inducingplasmids of Agrobacterium tumefaciens; the cauliflower mosaic virus(CaMV) 19S and 35S promoters; the enhanced CaMV 35S promoter; theFigwort Mosaic Virus (FMV) 35S promoter; the light-inducible promoterfrom the small subunit of ribulose-1,5-bisphosphate carboxylase(ssRUBISCO); the EIF-4A promoter from tobacco (Mandel et al., 1995);corn sucrose synthetase 1 (Yang and Russell, 1990); corn alcoholdehydrogenase 1 (Vogel et al., 1989); corn light harvesting complex(Simpson, 1986); corn heat shock protein (Odell et al., 1985); thechitinase promoter from Arabidopsis (Samac et al., 1991); the LTP (LipidTransfer Protein) promoters from broccoli (Pyee et al., 1995); petuniachalcone isomerase (Van Tunen et al., 1988); bean glycine rich protein 1(Keller et al., 1989); potato patatin (Wenzler et al., 1989); theubiquitin promoter from maize (Christensen et al., 1992); and the actinpromoter from rice (McElroy et al., 1990).

An additional promoter is preferably seed selective, tissue selective,constitutive, or inducible. The promoter is most preferably the nopalinesynthase (nos), octopine synthase (ocs), mannopine synthase (mas),cauliflower mosaic virus 19S and 35S (CaMV19S, CaMV35S), enhanced CaMV(eCaMV), ribulose 1,5-bisphosphate carboxylase (ssRUBISCO), figwortmosaic virus (FMV), CaMV derived AS4, tobacco RB7, wheat POX1, tobaccoEIF-4, lectin protein (Le1), or rice RC2 promoter.

Recombinant Vectors Having Additional Structural Nucleic Acid Sequences

The recombinant vector may also contain one or more additionalstructural nucleic acid sequences. These additional structural nucleicacid sequences may generally be any sequences suitable for use in arecombinant vector. Such structural nucleic acid sequences include,without limitation, any of the structural nucleic acid sequences, andmodified forms thereof, described above. The additional structuralnucleic acid sequences may also be operably linked to any of the abovedescribed promoters. The one or more structural nucleic acid sequencesmay each be operably linked to separate promoters. Alternatively, thestructural nucleic acid sequences may be operably linked to a singlepromoter (i.e., a single operon).

The additional structural nucleic acid sequences include, withoutlimitation, those encoding seed storage proteins, fatty acid pathwayenzymes, tocopherol biosynthetic enzymes, amino acid biosyntheticenzymes, and starch branching enzymes.

Preferred seed storage proteins include zeins (U.S. Pat. Nos. 4,886,878;4,885,357; 5,215,912; 5,589,616; 5,508,468; 5,939,599; 5,633,436; and5,990,384; Patent Applications WO 90/01869, WO 91/13993, WO 92/14822, WO93/08682, WO 94/20628, WO 97/28247, WO 98/26064, and WO 99/40209), 7Sproteins (U.S. Pat. Nos. 5,003,045 and 5,576,203), Brazil nut protein(U.S. Pat. No. 5,850,024), phenylalanine free proteins (PatentApplication WO 96/17064), albumin (Patent Application WO 97/35023),β-conglycinin (Patent Application WO 00/19839), 11S (U.S. Pat. No.6,107,051), α-hordothionin (U.S. Pat. Nos. 5,885,802 and 5,885,801),arcelin seed storage proteins (U.S. Pat. No. 5,270,200), lectins (U.S.Pat. No. 6,110,891), and glutenin (U.S. Pat. Nos. 5,990,389 and5,914,450).

Preferred fatty acid pathway enzymes include thioesterases (U.S. Pat.Nos. 5,512,482; 5,530,186; 5,945,585; 5,639,790; 5,807,893; 5,955,650;5,955,329; 5,759,829; 5,147,792; 5,304,481; 5,298,421; 5,344,771; and5,760,206), and desaturases (U.S. Pat. Nos. 5,689,050; 5,663,068;5,614,393; 5,856,157; 6,117,677; 6,043,411; 6,194,167; 5,705,391;5,663,068; 5,552,306; 6,075,183; 6,051,754; 5,689,050; 5,789,220;5,057,419; 5,654,402; 5,659,645; 6,100,091; 5,760,206; 6,172,106;5,952,544; 5,866,789; 5,443,974; and 5,093,249).

Preferred tocopherol biosynthetic enzymes include tyrA, slr1736, ATPT2,dxs, dxr, GGPPS, HPPD, GMT, MT1, tMT2, AANTJ, slr1737, and an antisenseconstruct for homogentisic acid dioxygenase (Kridl et al., Seed Sci.Res., 1:209-219 (1991); Keegstra, Cell, 56(2):247-53 (1989); Nawrath etal., Proc. Natl. Acad. Sci. (U.S.A.), 91:12760-12764 (1994); Xia et al.,J. Gen. Microbiol., 138:1309-1316 (1992); Cyanobasehttp://www.kazusa.or.jp/cyanobase; Lois et al., Proc. Natl. Acad. Sci.(U.S.A.), 95(5):2105-2110 (1998); Takahashi, et al. Proc. Natl. Acad.Sci. (U.S.A.), 95(17):9879-9884 (1998); Norris et al., Plant Physiol.,117:1317-1323 (1998); Bartley and Scolnik, Plant Physiol., 104:1469-1470(1994); Smith et al., Plant J., 11:83-92 (1997); WO 00/32757; WO00/10380; Saint Guily et al., Plant Physiol., 100(2):1069-1071 (1992);Sato et al., J. DNA Res., 7(1):31-63 (2000)).

Preferred amino acid biosynthetic enzymes include anthranilate synthase(U.S. Pat. No. 5,965,727; Patent Applications WO 97/26366, WO 99/11800,and WO 99/49058), tryptophan decarboxylase (Patent Application WO99/06581), threonine decarboxylase (U.S. Pat. Nos. 5,534,421 and5,942,660; Patent Application WO 95/19442), threonine deaminase (PatentApplications WO 99/02656 and WO 98/55601), and aspartate kinase (U.S.Pat. Nos. 5,367,110; 5,858,749; and 6,040,160).

Preferred starch branching enzymes include those set forth in U.S. Pat.Nos. 6,232,122 and 6,147,279; and Patent Application WO 97/22703.

Alternatively, the second structural nucleic acid sequence may bedesigned to down-regulate a specific nucleic acid sequence. This istypically accomplished by operably linking the second structural aminoacid, in an antisense orientation, with a promoter. One of ordinaryskill in the art is familiar with such antisense technology. Any nucleicacid sequence may be negatively regulated in this manner. Preferabletarget nucleic acid sequences contain a low content of essential aminoacids, yet are expressed at relatively high levels in particulartissues. For example, β-conglycinin and glycinin are expressedabundantly in seeds, but are nutritionally deficient with respect toessential amino acids. This antisense approach may also be used toeffectively remove other undesirable proteins, such as antifeedants(e.g., lectins), albumin, and allergens, from plant-derived foodstuffs,or to down-regulate catabolic enzymes involved in degradation of desiredcompounds such as essential amino acids.

Selectable Markers

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to: a neo gene (Potrykus et al., 1985), which codes forkanamycin resistance and can be selected for using kanamycin, RptII,G418, hpt etc.; a bar gene which codes for bialaphos resistance; amutant EPSP synthase gene (Hinchee et al., 1988; Reynaerts et al.,1988), aadA (Jones et al., 1987) which encodes glyphosate resistance; anitrilase gene which confers resistance to bromoxynil (Stalker et al.,1988); a mutant acetolactate synthase gene (ALS) which confersimidazolinone or sulphonylurea resistance (EP 154 204, 1985), ALS(D'Halluin et al., 1992), and a methotrexate resistant DHFR gene(Thillet et al., 1988). The selectable marker is preferably anantibiotic resistance coding sequence, or an herbicide (e.g.,glyphosate) resistance coding sequence. The selectable marker is mostpreferably a kanamycin, hygromycin, or herbicide resistance marker.

A vector or construct may also include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude: a β-glucuronidase or uidA gene (GUS) which encodes an enzymefor which various chromogenic substrates are known (Jefferson, 1987));an R-locus gene, which encodes a product that regulates the productionof anthocyanin pigments (red color) in plant tissues (Dellaporta et al.,1988); a β-lactamase gene (Sutcliffe et al., 1978), a gene which encodesan enzyme for which various chromogenic substrates are known (e.g.,PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al.,1986); a xylE gene (Zukowsky et al., 1983) which encodes a catecholdioxygenase that can convert chromogenic catechols; an α-amylase gene(Ikatu et al., 1990); a tyrosinase gene (Katz et al., 1983) whichencodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinonewhich in turn condenses to melanin; an α-galactosidase, which will turna chromogenic a-galactose substrate.

Included within the term or phrase “selectable or screenable markergenes” are also genes which encode a secretable marker whose secretioncan be detected as a means of identifying or selecting for transformedcells. Examples include markers which encode a secretable antigen thatcan be identified by antibody interaction, or even secretable enzymeswhich can be detected catalytically. Secretable proteins fall into anumber of classes, including small, diffusible proteins which aredetectable, (e.g., by ELISA), small active enzymes which are detectablein extracellular solution (e.g., α-amylase, β-lactamase,phosphinothricin transferase), or proteins which are inserted or trappedin the cell wall (such as proteins which include a leader sequence suchas that found in the expression unit of extension or tobacco PR-S).Other possible selectable and/or screenable marker genes will beapparent to those of skill in the art.

Other Elements in the Recombinant Vector

Various cis-acting untranslated 5′ and 3′ regulatory sequences may beincluded in the recombinant nucleic acid vector. Any such regulatorysequences may be provided in a recombinant vector with other regulatorysequences. Such combinations can be designed or modified to producedesirable regulatory features.

A 3′ non-translated region typically provides a transcriptionaltermination signal, and a polyadenylation signal which functions inplants to cause the addition of adenylate nucleotides to the 3′ end ofthe mRNA. These may be obtained from the 3′ regions of the nopalinesynthase (nos) coding sequence, a soybean 7Sα′ storage protein codingsequence, the arcelin-5 coding sequence, the albumin coding sequence,and the pea ssRUBISCO E9 coding sequence. Typically, nucleic acidsequences located a few hundred base pairs downstream of thepolyadenylation site serve to terminate transcription. These regions arerequired for efficient polyadenylation of transcribed mRNA.

Translational enhancers may also be incorporated as part of therecombinant vector. Thus the recombinant vector may preferably containone or more 5′ non-translated leader sequences which serve to enhanceexpression of the nucleic acid sequence. Such enhancer sequences may bedesirable to increase or alter the translational efficiency of theresultant mRNA. Preferred 5′ nucleic acid sequences include dSSU 5′,PetHSP70 5′, and GmHSP17.9 5′ (U.S. Pat. No. 5,362,865).

The recombinant vector may further comprise a nucleic acid sequenceencoding a transit peptide. This peptide may be useful for directing aprotein to the extracellular space, a plastid, or to some othercompartment inside or outside of the cell. (see, e.g., EP 0 218 571,U.S. Pat. Nos. 4,940,835; 5,88,624; 5,610,041; 5,618,988; and6,107,060).

The structural nucleic acid sequence in the recombinant vector maycomprise introns. The introns may be heterologous with respect to thestructural nucleic acid sequence. Preferred introns include the riceactin intron and the corn HSP70 intron for monocotyledon plants and pearbcS3A intron 1 and petunia SSU301 introns for dicotyledon plants.

Fusion Proteins

Any of the above described structural nucleic acid sequences, andmodified forms thereof, may be linked with additional nucleic acidsequences to encode fusion proteins. The additional nucleic acidsequence preferably encodes at least 1 amino acid, peptide, or protein.Many possible fusion combinations exist.

For instance, the fusion protein may provide a “tagged” epitope tofacilitate detection of the fusion protein, such as GST, GFP, FLAG, orpolyHIS. Such fusions preferably encode between 1 and 50 amino acids,more preferably between 5 and 30 additional amino acids, and even morepreferably between 5 and 20 amino acids.

Alternatively, the fusion may provide regulatory, enzymatic, cellsignaling, or intercellular transport functions. For example, a sequenceencoding a plastid transit peptide may be added to direct a fusionprotein to the chloroplasts within seeds. Such fusion partnerspreferably encode between 1 and 1000 additional amino acids, morepreferably between 5 and 500 additional amino acids, and even morepreferably between 10 and 250 amino acids.

Sequence Analysis

In the present invention, sequence similarity or identity is preferablydetermined using the “Best Fit” or “Gap” programs of the SequenceAnalysis Software Package™ (Version 10; Genetics Computer Group, Inc.,University of Wisconsin Biotechnology Center, Madison, Wis.). “Gap”utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch,1970) to find the alignment of two sequences that maximizes the numberof matches and minimizes the number of gaps. “BestFit” performs anoptimal alignment of the best segment of similarity between twosequences. Optimal alignments are found by inserting gaps to maximizethe number of matches using the local homology algorithm of Smith andWaterman (Smith and Waterman, 1981; Smith et al., 1983).

The Sequence Analysis Software Package described above contains a numberof other useful sequence analysis tools for identifying homologues ofthe presently disclosed nucleotide and amino acid sequences. Forexample, the “BLAST” program searches for sequences similar to a querysequence (either peptide or nucleic acid) in a specified database (e.g.,sequence databases maintained at the National Center for BiotechnologyInformation (NCBI) in Bethesda, Md.); “FastA” (Lipman and Pearson, 1985;see also, Pearson and Lipman, 1988; Pearson, 1990) performs a Pearsonand Lipman search for similarity between a query sequence and a group ofsequences of the same type (nucleic acid or protein); “TfastA” performsa Pearson and Lipman search for similarity between a protein querysequence and any group of nucleotide sequences (it translates thenucleotide sequences in all six reading frames before performing thecomparison); “FastX” performs a Pearson and Lipman search for similaritybetween a nucleotide query sequence and a group of protein sequences,taking frameshifts into account. “TfastX” performs a Pearson and Lipmansearch for similarity between a protein query sequence and any group ofnucleotide sequences, taking frameshifts into account (it translatesboth strands of the nucleic acid sequence before performing thecomparison).

Probes and Primers

Short nucleic acid sequences having the ability to specificallyhybridize to complementary nucleic acid sequences may be produced andutilized in the present invention. Such short nucleic acid molecules maybe used as probes to identify the presence of a complementary nucleicacid sequence in a given sample. Thus, by constructing a nucleic acidprobe which is complementary to a small portion of a particular nucleicacid sequence, the presence of that nucleic acid sequence may bedetected and assessed.

Alternatively, the short nucleic acid sequences may be used asoligonucleotide primers to amplify or mutate a complementary nucleicacid sequence using PCR technology. These primers may also facilitatethe amplification of related complementary nucleic acid sequences (e.g.,related nucleic acid sequences from other species).

Short nucleic acid sequences may be used as primers and specifically asPCR primers. A PCR probe is a nucleic acid molecule capable ofinitiating a polymerase activity while in a double-stranded structurewith another nucleic acid. Various methods for determining the structureof PCR primers and PCR techniques exist in the art. Computer generatedsearches using programs such as Primer3(www.genome.wi.mitedu/cgi-bin/primer/primer3.cgi), STSPipeline(www-genome.wi.mit.edu/cgi-bin/www.STS_Pipeline), or GeneUp (Pesole etal., 1998), for example, can be used to identify potential PCR primers.

Any of the nucleic acid sequences disclosed herein may be used as aprimer or probe. Use of these probes or primers may greatly facilitatethe identification of transgenic plants which contain the presentlydisclosed promoters and structural nucleic acid sequences. Such probesor primers may also be used to screen cDNA or genomic libraries foradditional nucleic acid sequences related to or sharing homology withthe presently disclosed promoters and structural nucleic acid sequences.

A primer or probe is generally complementary to a portion of a nucleicacid sequence that is to be identified, amplified, or mutated and ofsufficient length to form a stable and sequence-specific duplex moleculewith its complement. The primer or probe preferably is about 10 to about200 nucleotides long, more preferably is about 10 to about 100nucleotides long, even more preferably is about 10 to about 50nucleotides long, and most preferably is about 14 to about 30nucleotides long.

The primer or probe may, for example without limitation, be prepared bydirect chemical synthesis, by PCR (U.S. Pat. Nos. 4,683,195 and4,683,202), or by excising the nucleic acid specific fragment from alarger nucleic acid molecule.

Transgenic Plants and Transformed Plant Host Cells

The invention is also directed to transgenic plants and transformed hostcells which comprise a promoter operably linked to a heterologousstructural nucleic acid sequence. Other nucleic acid sequences may alsobe introduced into the plant or host cell along with the promoter andstructural nucleic acid sequence. These other sequences may include 3′transcriptional terminators, 3′ polyadenylation signals, otheruntranslated nucleic acid sequences, transit or targeting sequences,selectable markers, enhancers, and operators. Preferred nucleic acidsequences of the present invention, including recombinant vectors,structural nucleic acid sequences, promoters, and other regulatoryelements, are described above.

In a further embodiment of the present invention, any of the promotersequences described herein can be used to express genes that improve theoverall seedling vigor of a plant. In a preferred embodiment, a plant ofthe present invention that has improved seedling vigor comprises SEQ IDNOs: 1 or 2. As used herein, a seedling having “improved overallseedling vigor” means a seedling that has improved vigor relative to aplant with a similar genetic background that lacks a promoter sequenceof the present invention.

In a further embodiment of the present invention, any of the promotersequences described herein can be used to express genes that improve thegrowth rate of a seedling plant. In a preferred embodiment, a plant ofthe present invention that has improved seedling growth rate comprisesSEQ ID NOs: 1 or 2. As used herein, a seedling having “improved growthrate” means a seedling that has grows at a faster pace relative to aplant with a similar genetic background at the same stage of growth thatlacks a promoter sequence of the present invention.

In a further embodiment of the present invention, any of the promotersequences described herein can be used to express genes that lengthenthe period during which a plant fills its seeds. In a preferredembodiment, seed fill comprises pod fill in soybean plants or kernelfilling in corn.

In another embodiment of the present invention, any of the promotersequences described herein can be used to express genes that alter thesink strength regulation of a plant in order to enhance the rate ofgrain fill in seed. As used herein, to “alter” sink strength regulationof a plant means to increase or decrease the sink strength regulation ofa plant relative to a plant having a similar genetic background thatlacks a promoter of the present invention.

In yet another embodiment of the present invention, any of the promotersequences described herein can be used to express genes that regulate orenhance seed viability or seed storage, thereby improving stand count oryield or both.

In a preferred embodiment, the transgenic plants and transformed hostcells comprise a seed promoter. In a most preferred embodiment, thetransgenic plants and transformed host cells comprise any nucleic acidmolecule of the present invention as described herein, including anucleic acid sequence selected from the group consisting of SEQ ID NOs:1-4, and complements thereof.

In a particularly preferred embodiment, the transgenic plant of thepresent invention is a soybean plant. In a preferred embodiment, asoybean plant of the present invention comprises one or more introducednucleic acid molecules of the present invention. In a preferredembodiment, a transformed soybean plant of the present inventioncomprises a nucleic acid molecule selected from the group consisting ofSEQ ID NOs: 1-4. In a preferred embodiment a transformed soybean plantof the present invention comprises a nucleic acid molecule comprisingSEQ ID NO: 1.

Means for preparing such recombinant vectors are well known in the art.For example, methods for making recombinant vectors particularly suitedto plant transformation are described in U.S. Pat. Nos. 4,971,908;4,940,835; 4,769,061; and 4,757,011. These vectors have also beenreviewed (Rodriguez et al., 1988; Glick et al., 1993).

Typical vectors useful for expression of nucleic acids in cells andhigher plants are well known in the art and include vectors derived fromthe tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers etal., 1987). Other recombinant vectors useful for plant transformationhave also been described (Fromm et al., 1985). Elements of suchrecombinant vectors include, without limitation, those discussed above.

A transformed host cell may generally be any cell that is compatiblewith the present invention. A transformed host plant or cell can be orderived from a monocotyledonous plant or a dicotyledonous plantincluding, but not limited to canola, crambe, maize, mustard, castorbean, sesame, cottonseed, linseed, soybean, Arabidopsis phaseolus,peanut, alfalfa, wheat, rice, oat, sorghum, rapeseed, rye, tritordeum,millet, fescue, perennial ryegrass, sugarcane, cranberry, papaya,banana, safflower, oil palms, flax, muskmelon, apple, cucumber,dendrobium, gladiolus, chrysanthemum, liliacea, cotton, eucalyptus,sunflower, Brassica campestris, Brassica napus, turfgrass, sugarbeet,coffee and dioscorea (Christou, In: Particle Bombardment for GeneticEngineering of Plants, Biotechnology Intelligence Unit, Academic Press,San Diego, Calif. (1996)), with canola, maize, Brassica campestris,Brassica napus, rapeseed, soybean, safflower, wheat, rice and sunflowerpreferred, and canola, rapeseed, maize, Brassica campestris, Brassicanapus, soybean, sunflower, safflower, oil palms, and peanut morepreferred. In a particularly preferred embodiment, the plant or cell isor derived from canola. In another particularly preferred embodiment,the plant or cell is or derived from Brassica napus. In anotherparticularly preferred embodiment, the plant or cell is or derived fromsoybean.

The soybean cell or plant is preferably an elite soybean cell line. An“elite line” is any commercially available line that has resulted frombreeding and selection for superior agronomic performance. Examples ofelite lines, include without limitation, the following: HARTZ™ varietyH4994, HARTZ™ variety H5218, HARTZ™ variety H5350, HARTZ™ variety H5545,HARTZ™ variety H5050, HARTZ™ variety H5454, HARTZ™ variety H5233, HARTZ™variety H5488, HARTZ™ variety HLA572, HARTZ™ variety H6200, HARTZ™variety H6104, HARTZ™ variety H6255, HARTZ™ variety H6586, HARTZ™variety H6191, HARTZ™ variety H7440, HARTZ™ variety H4452 RoundupReady™, HARTZ™ variety H4994 Roundup Ready™, HARTZ™ variety H4988Roundup Ready™, HARTZ™ variety H5000 Roundup Ready™, HARTZ™ varietyH5147 Roundup Ready™, HARTZ™ variety H5247 Roundup Ready™, HARTZ™variety H5350 Roundup Ready™, HARTZ™ variety H5545 Roundup Ready™,HARTZ™ variety H5855 Roundup Ready™, HARTZ™ variety H5088 RoundupReady™, HARTZ™ variety H5164 Roundup Ready™, HARTZ™ variety H5361Roundup Ready™, HARTZ™ variety H5566 Roundup Ready™, HARTZ™ varietyH5181 Roundup Ready™, HARTZ™ variety H5889 Roundup Ready™, HARTZ™variety H5999 Roundup Ready™, HARTZ™ variety H6013 Roundup Ready™,HARTZ™ variety H6255 Roundup Ready™, HARTZ™ variety H6454 RoundupReady™, HARTZ™ variety H6686 Roundup Ready™, HARTZ™ variety H7152Roundup Ready™, HARTZ™ variety H7550 Roundup Ready™, HARTZ™ varietyH8001 Roundup Ready™ (HARTZ SEED, Stuttgart, AR); A0868, AG0901, A1553,A1900, AG1901, A1923, A2069, AG2101, AG2201, A2247, AG2301, A2304,A2396, AG2401, AG2501, A2506, A2553, AG2701, AG2702, A2704, A2833,A2869, AG2901, AG2902, AG3001, AG3002, A3204, A3237, A3244, AG3301,AG3302, A3404, A3469, AG3502, A3559, AG3601, AG3701, AG3704, AG3750,A3834, AG3901, A3904, A4045 AG4301, A4341, AG4401, AG4501, AG4601,AG4602, A4604, AG4702, AG4901, A4922, AG5401, A5547, AG5602, A5704,AG5801, AG5901, A5944, A5959, AG6101, QR4459 and QP4544 (Asgrow Seeds,Des Moines, Iowa); DeKalb variety CX445 (DeKalb, Ill.).

The invention is also directed to a method of producing transformedplants which comprise, in a 5′ to 3′ orientation, a promoter operablylinked to a heterologous structural nucleic acid sequence. Othersequences may also be introduced into plants along with the promoter andstructural nucleic acid sequence. These other sequences may include 3′transcriptional terminators, 3′ polyadenylation signals, otheruntranslated sequences, transit or targeting sequences, selectablemarkers, enhancers, and operators. Preferred recombinant vectors,structural nucleic acid sequences, promoters, and other regulatoryelements including, without limitation, those described herein.

The method generally comprises the steps of selecting a suitable plant,transforming the plant with a recombinant vector, and obtaining thetransformed host cell.

There are many methods for introducing nucleic acids into plants.Suitable methods include bacterial infection (e.g., Agrobacterium),binary bacterial artificial chromosome vectors, direct delivery ofnucleic acids (e.g., via PEG-mediated transformation,desiccation/inhibition-mediated nucleic acid uptake, electroporation,agitation with silicon carbide fibers, and acceleration of nucleic acidcoated particles, etc. (reviewed in Potrykus et al., 1991)).

Technology for introduction of nucleic acids into cells is well known tothose of skill in the art. Methods can generally be classified into fourcategories: (1) chemical methods (Graham and van der Eb, 1973; Zatloukalet al., 1992); (2) physical methods such as microinjection (Capecchi,1980), electroporation (Wong and Neumann, 1982; Fromm et al., 1985; U.S.Pat. No. 5,384,253), and particle acceleration (Johnston and Tang, 1994;Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al., 1993;Eglitis and Anderson, 1988); and (4) receptor-mediated mechanisms(Curiel et al., 1992; Wagner et al., 1992). Alternatively, nucleic acidscan be directly introduced into pollen by directly injecting a plant'sreproductive organs (Zhou et al., 1983; Hess, 1987; Luo et al., 1988;Pena et al., 1987). In another aspect nucleic acids may also be injectedinto immature embryos (Neuhaus et al., 1987).

Regeneration, development, and cultivation of plants from transformedplant protoplast or explants is taught in the art (Weissbach andWeissbach, 1988; Horsch et al., 1985). Transformants are generallycultured in the presence of a selective media which selects for thesuccessfully transformed cells and induces the regeneration of plantshoots (Fraley et al., 1983). Such shoots are typically obtained withintwo to four months.

Shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Many of the shoots will develop roots. These are thentransplanted to soil or other media to allow the continued developmentof roots. The method, as outlined, will generally vary depending on theparticular plant employed.

Preferably, the regenerated transgenic plants are self-pollinated toprovide homozygous transgenic plants. Alternatively, pollen obtainedfrom the regenerated transgenic plants may be crossed withnon-transgenic plants, preferably inbred lines of agronomicallyimportant species. Conversely, pollen from non-transgenic plants may beused to pollinate the regenerated transgenic plants.

A transgenic plant may pass along the nucleic acid sequence encoding theenhanced gene expression to its progeny. The transgenic plant ispreferably homozygous for the nucleic acid encoding the enhanced geneexpression and transmits that sequence to all of its offspring upon as aresult of sexual reproduction. Progeny may be grown from seeds producedby the transgenic plant. These additional plants may then beself-pollinated to generate a true breeding line of plants.

The progeny from these plants are evaluated, among other things, forgene expression. The gene expression may be detected by several commonmethods such as western blotting, northern blotting,immunoprecipitation, and ELISA.

Plants or agents of the present invention can be utilized in methods,for example without limitation, to obtain a seed that expresses astructural nucleic acid molecule in that seed, to obtain a seed enhancedin a product of a structural gene, to obtain meal enhanced in a productof a structural gene, to obtain feedstock enhanced in a product of astructural gene, and to obtain oil enhanced in a product of a structuralgene

Plants utilized in such methods may be processed. A plant or plant partmay be separated or isolated from other plant parts. A preferred plantpart for this purpose is a seed. It is understood that even afterseparation or isolation from other plant parts, the isolated orseparated plant part may be contaminated with other plant parts. In apreferred aspect, the separated plant part is greater than 50% (w/w) ofthe separated material, more preferably, greater than 75% (w/w) of theseparated material, and even more preferably greater than 90% (w/w) ofthe separated material. Plants or plant parts of the present inventiongenerated by such methods may be processed into products using knowntechniques. Preferred products are meal, feedstock and oil.

Feed, Meal, Protein and Oil Preparations

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, protein or oil preparation. Aparticularly preferred plant part for this purpose is a seed. In apreferred embodiment the feed, meal, protein or oil preparation isdesigned for ruminant animals. Methods to produce feed, meal, proteinand oil preparations are known in the art. See, for example, U.S. Pat.Nos. 4,957,748; 5,100,679; 5,219,596; 5,936,069; 6,005,076; 6,146,669;and 6,156,227. In a preferred embodiment, the protein preparation is ahigh protein preparation. Such a high protein preparation preferably hasa protein content of greater than 5% w/v, more preferably 10% w/v, andeven more preferably 15% w/v. In a preferred oil preparation, the oilpreparation is a high oil preparation with an oil content derived from aplant or part thereof of the present invention of greater than about 5%w/v, more preferably greater than about 10% w/v, and even morepreferably greater than about 15% w/v. In a preferred embodiment the oilpreparation is a liquid and of a volume greater than about 1, about 5,about 10, or about 50 liters. The present invention provides for oilproduced from plants of the present invention or generated by a methodof the present invention. Such oil may be a minor or major component ofany resultant product. Moreover, such oil may be blended with otheroils. In a preferred embodiment, the oil produced from plants of thepresent invention or generated by a method of the present inventionconstitutes greater than about 0.5%, about 1%, about 5%, about 10%,about 25%, about 50%, about 75%, or about 90% by volume or weight of theoil component of any product. In another embodiment, the oil preparationmay be blended and can constitute greater than about 10%, about 25%,about 35%, about 50%, or about 75% of the blend by volume. Oil producedfrom a plant of the present invention can be admixed with one or moreorganic solvents or petroleum distillates.

In a further embodiment, meal of the present invention may be blendedwith other meals. In a preferred embodiment, the meal produced fromplants of the present invention or generated by a method of the presentinvention constitutes greater than about 0.5%, about 1%, about 5%, about10%, about 25%, about 50%, about 75%, or about 90% by volume or weightof the meal component of any product. In another embodiment, the mealpreparation may be blended and can constitute greater than about 10%,about 25%, about 35%, about 50%, or about 75% of the blend by volume.

Seed Containers

Seeds of the plants may be placed into a container. As used herein, acontainer is any object capable of holding such seeds. A containerpreferably contains greater than about 500, about 1,000, about 5,000, orabout 25,000 seeds where at least about 10%, about 25%, about 50%, about75%, or about 100% of the seeds are derived from a plant of the presentinvention.

Breeding Programs

Plants of the present invention can be part of or generated from abreeding program. The choice of breeding method depends on the mode ofplant reproduction, the heritability of the trait(s) being improved, andthe type of cultivar used commercially (e.g., F₁ hybrid cultivar,pureline cultivar, etc). Selected, non-limiting approaches, for breedingthe plants of the present invention are set forth below. A breedingprogram can be enhanced using marker assisted selection of the progenyof any cross. It is further understood that any commercial andnon-commercial cultivars can be utilized in a breeding program. Factorssuch as, for example, emergence vigor, vegetative vigor, stresstolerance, disease resistance, branching, flowering, seed set, seedsize, seed density, standability, and threshability and the like willgenerally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredembodiment a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated selfing and selection, producing many new genetic combinations.

The development of new cultivars requires the development and selectionof varieties, the crossing of these varieties and the selection ofsuperior hybrid crosses. The hybrid seed can be produced by manualcrosses between selected male-fertile parents or by using male sterilitysystems. Hybrids are selected for certain single gene traits such as podcolor, flower color, seed yield, pubescence color, or herbicideresistance, which indicate that the seed is truly a hybrid. Additionaldata on parental lines, as well as the phenotype of the hybrid,influence the breeder's decision whether to continue with the specifichybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. An F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals from thebest families is carried out. Replicated testing of families can beginin the F₄ generation to improve the effectiveness of selection fortraits with low heritability. At an advanced stage of inbreeding (i.e.,F₆ and F₇), the best lines or mixtures of phenotypically similar linesare tested for potential release as new cultivars.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or more podsfrom each plant in a population and thresh them together to form a bulk.Part of the bulk is used to plant the next generation and part is put inreserve. The procedure has been referred to as modified single-seeddescent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seed of apopulation each generation of inbreeding.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Fehr, Principles of Cultivar Development, Vol. 1, pp. 2-3(1987)).

A transgenic plant of the present invention may also be reproduced usingapomixis. Apomixis is a genetically controlled method of reproduction inplants where the embryo is formed without union of an egg and a sperm.There are three basic types of apomictic reproduction: 1) apospory wherethe embryo develops from a chromosomally unreduced egg in an embryo sacderived from the nucleus, 2) diplospory where the embryo develops froman unreduced egg in an embryo sac derived from the megaspore mothercell, and 3) adventitious embryony where the embryo develops directlyfrom a somatic cell. In most forms of apomixis, pseudogamy orfertilization of the polar nuclei to produce endosperm is necessary forseed viability. In apospory, a nurse cultivar can be used as a pollensource for endosperm formation in seeds. The nurse cultivar does notaffect the genetics of the aposporous apomictic cultivar since theunreduced egg of the cultivar develops parthenogenetically, but makespossible endosperm production. Apomixis is economically important,especially in transgenic plants, because it causes any genotype, nomatter how heterozygous, to breed true. Thus, with apomicticreproduction, heterozygous transgenic plants can maintain their geneticfidelity throughout repeated life cycles. Methods for the production ofapomictic plants are known in the art. See, U.S. Pat. No. 5,811,636.

EXAMPLES

The following examples are provided and should not be interpreted in anyway to limit the scope of the present invention.

Example 1

The promoter for the soybean lea9 gene (Hsing, et al., Plant Physiol.,100:2121-2122 (1992)) is PCR amplified from soybean genomic DNA (cv.Asgrow 4922) using the following primers based on the publishedsequence:

Primer ID lea9-5′ (SEQ ID NO: 5)5′-ACCTGCGGCCGCCAAGTACTTACGCCACACCAACTTAC-3′; and Primer ID lea9-3′ (SEQID NO: 6) 5′-GCAGCTGTTTCCTTGATGGACTCTC-3′.

All oligonucleotide primers are obtained from Gibco Life Technologies(Grand Island, N.Y.). The initial PCR reaction is performed using theTaq DNA polymerase kit (Boehringer Mannheim, Germany). The nested PCRreaction follows the primary PCR reaction, using the 5′ primer of theinitial reaction and lea9-3‘nest’ (GAAGATCTCCTGCAATTTCAAAGATCAATTATTTCC) (SEQ ID NO: 7). The following tables summarize thecomponents used for these reactions:

Initial PCR Reaction

Component Amount Soybean Genomic DNA (80 ng/μl) 1.0 μl dNTP mix (10 mMof each dNTP) 1.0 μl Primer lea9-5′ (10 μM) 1.0 μl Primer lea9-3′ (10μM) 1.0 μl 10X PCR Buffer (containing MgCl₂)   5 μl (final conc of 1X)Taq Polymerase 1.0 μl Distilled Water bring to 50 μl final volume

Nested PCR Reactions

Component Amount aliquot from primary PCR reaction 1.0 μl dNTP mix (10mM of each dNTP) 1.0 μl Primer lea9-5′ (10 μM) 1.0 μl Primerlea9-3′nest′ (10 μM) 1.0 μl 10X PCR Buffer (containing MgCl₂)   5 μl(final conc of 1X) Taq polymerase 1.0 μl Distilled Water bring to 50 μlfinal volume

The reactions are heated to 95° C. before adding the polymerase enzyme.Both the initial and nested PCR reactions are initiated by denaturingthe sample at 94° C. for 2 minutes. The reaction mixture is incubatedfor 7 cycles consisting of 94° C. for 30 seconds, 72° C. for 2.5 minutes(−1° C./cycle). The reaction mixture is then incubated for 30 cyclesconsisting of 94° C. for 30 seconds, 68° C. for 2 minutes, 72° C. for 2seconds minutes with a final step of 72° C. for 10 minutes. Thereactions are held at 4° C. until next step.

The product from the nested PCR reaction is purified by agarose gelelectrophoresis using the Qiagen Gel Extraction Kit (Qiagen, Valencia,Calif.; product Cat#28704). An aliquot of the purified PCR product isthen digested with the restriction enzymes NotI and BglII (Promega,Madison, Wis.) and ligated into a pMON8677 (FIG. 1) backbone, which isalso cut with NotI and BglII, to remove the e35S promoter cassette. Theligation is performed with the Rapid DNA Ligation Kit fromBoehringer-Mannheim (Germany, catalog number Cat#1635379) according tothe manufacturer's recommendations. The resulting construct in which thelea9 promoter is directly upstream of the uidA (β-glucuronidase)reporter gene is named pMON49801 (FIG. 2) and is used in transient assayanalysis.

An aliquot of the ligation reaction is transformed into a suitable E.coli (DH5α) and the cells are plated on selection medium consisting ofLuria Broth ((LB) 10% Bactotryptone, 5% yeast extract, and 10% NaCl),with 100 μg/ml ampicillin. Bacterial transformants are selected, grownin liquid culture, and the plasmid DNA is isolated using the QiaprepSpin Microprep Kit (Qiagen Corp., Valencia, Calif.). Purified plasmidcontaining the predicted insert size based on restriction enzymeanalysis is sequenced.

Example 2

The promoter for the Arabidopsis thaliana per1 gene (Haslekas, et al.,Plant Mol. Biol. 36:833-845 (1998)) is PCR amplified from Arabidopsis(cv. Columbia) genomic DNA using the following primers designed from thepublished gene sequence:

Primer ID JA44 5′-GGATCCAAATCAAAGTTTAATAGACTT-3′; (SEQ ID NO: 8) andPrimer ID JA46 5′-TCTAGGTTGGGGACCGTGTCTC-3′. (SEQ ID NO: 9)

The oligonucleotide primers are supplied by Gibco Life Technologies(Grand Island, N.Y.). The PCR is performed with the Expand High FidelityPCR System (Boehringer Mannheim, (Germany), catalog number 1732641).Following the primary PCR reaction, a second reaction using nested PCRprimers is performed using 1 μl of product from the primary reaction andthe following primers designed from the published sequence:

Primer ID JA45 (SEQ ID NO: 10) 5′-TAGCGGCCGCTAATAGACTTTGCACCTCCAT-3′;and Primer ID JA47 (SEQ ID NO: 11)5′-AACCATGGTTTACCTTTTATATTTATATATAGAA-3′.The PCR components and conditions are outlined below:Primary PCR

Component Amount Arabidopsis Genomic DNA  0.5 μl (0.75ug) dNTP mix (10mM of each dNTP)  2.0 μl Primer JA44 (10 μM)  3.0 μl Primer JA46 (10 μM) 3.0 μl 10X PCR Buffer (containing MgCl₂)   10 μl (final conc of 1X)Polymerase enzyme 0.75 μl Distilled Water bring to 100 μl final volumeNested PCR

Component Amount aliquot from primary PCR reaction  1.0 μl dNTP mix (10mM of each dNTP)  2.0 μl Primer JA45 (10 μM)  3.0 μl Primer JA47 (10 μM) 3.0 μl 10X PCR Buffer (containing MgCl₂)   10 μl (final conc of 1X)Polymerase enzyme 0.75 μl Distilled Water bring to 100 μl final volume

The reactions are heated to 95° C. before adding the polymerase enzyme.Both the initial and nested PCR reactions are initiated by denaturingthe sample at 94° C. for 2 minutes. The reaction mixture is incubatedfor 10 cycles consisting of 94° C. for 15 seconds, 60° C. for 30 secondsand 72° C. for 90 seconds. The reaction mixture is then incubated for 26cycles consisting of 94° C. for 15 seconds, 60° C. for 30 seconds, 72°C. for 30 seconds for the initial cycle and 50 seconds for eachadditional cycle with a final step of 72° C. for 7 minutes. Thereactions are held at 4° C. until next step.

A 25 μl aliquot of the PCR product is taken for agarose gel analysis.The remaining PCR product is purified using the Qiagen PCR PurificationKit (Qiagen, Inc., Valencia, Calif., product number 28104) following theconditions recommended by the manufacturer. An aliquot of the purifiedPCR product is then digested with restriction enzymes NotI and NcoI(Promega, Madison, Wis.) and ligated into a pMON8677 backbone (FIG. 1),which had been cut with Nod and NcoI to remove the e35S promotercassette. The ligation is performed with the Rapid DNA Ligation Kit fromBoehringer-Mannheim (Germany, catalog number 1635379) according to themanufacturer's recommendations. The resulting construct is namedpMON42316 (FIG. 3) in which the AtPer1 promoter is directly upstream ofthe uidA (β-glucuronidase) reporter gene.

An aliquot of the ligation reaction is transformed into a suitable E.coli host (DH5α) and the cells plated on selection medium (LB with 100ug/ml carbenicillin). Bacterial transformants are selected, grown inliquid culture (LB with 100 ug/ml carbenicillin), and the plasmid DNA isisolated using the Qiaprep Spin Microprep Kit (Qiagen Corp., Valencia,Calif.). Purified plasmid containing the predicted insert size based onrestriction enzyme analysis are sequenced using primers JA45, JA47, anda primer named GUS5′-2, 5′-GTAACGCGCTTTCCCACCAACGCT (SEQ ID NO: 12),which anneals in the GUS coding region of the plasmid. The sequence ofthe cloned AtPer1 promoter matches that of the published promotersequence.

Example 3

The promoter for the soybean Sle2 gene (Calvo, et al., Theor. Appl.Genet., 94:957-967 (1997)) is amplified from the a soybean genomic DNAlibrary (cv. Williams 82, purchased from Stratagene, La Jolla, Calif.;cat. number 946103). The following primers, which are designed from thepublished gene sequence, are used for the primary reaction:

Primer ID JA41 5′-GTGTTACATTATCACTTATCCTGGTC-3′; (SEQ ID NO: 13) andPrimer ID Jeahre2 5′-GCTCAATTAACCCTCACTAAAGGGA-3′. (SEQ ID NO: 14)

The oligonucleotide primers are supplied by Gibco Life Technologies(Grand Island, N.Y.). The primary PCR reaction is performed with theExpand Long Template PCR system (Boehringer Mannheim, Germany, catalognumber 1681834).

Following the primary PCR reaction, a nested PCR reaction is performedusing 1 μl of product from the primary reaction as template DNA and thefollowing nested primers designed from the published sequence:

Primer ID JA43 5′-CTCTTGAGCACGTTCTTCTCCT-3′; (SEQ ID NO: 15) and PrimerID Jeahre2 5′-GCTCAATTAACCCTCACTAAAGGGA-3′. (SEQ ID NO: 16)

The PCR components and conditions for both reactions are outlined below:

Primary PCR (Long Template PCR Kit)

Component Amount Soybean Genomic DNA  1.0 μl dNTP mix (10 mM of eachdNTP)  2.5 μl Primer JA41 (10 μM)  1.0 μl Primer Jeahre2 (10 μM)  1.0 μl10X PCR Buffer #3 (containing MgCl₂)   5 μl (final conc of 1X)Polymerase enzyme mix 0.75 μl Distilled Water bring to 50 μl finalvolumeNested PCR (Long Template PCR Kit)

Component Amount aliquot from primary PCR reaction  1.0 μl dNTP mix (10mM of each dNTP)  2.5 μl Primer JA43 (10 μM)  1.0 μl Primer Jeahre2 (10μM)  1.0 μl 10X PCR Buffer #3(containing MgCl₂)   5 μl (final conc of1X) Polymerase enzyme mix 0.75 μl Distilled Water bring to 50 μl finalvolume

Both the primary and nested PCR reactions are initiated by denaturingthe sample at 94° C. for 2 minutes. The reaction mixture is incubatedfor 10 cycles consisting of 94° C. for 10 seconds, 69° C. for 30 seconds(−1.5° C./cycle), and 68° C. for 12 minutes. The reaction mixture isthen incubated for 25 cycles consisting of 94° C. for 10 seconds, 58° C.for 30 seconds, 68° C. for 12 minutes, and a final step of 68° C. for 7minutes. The reactions are held at 4° C. until the next step. Aftersuccessful cloning and identification of the Sle2 promoter region, thepromoter is re-amplified with the Expand High Fidelity PCR System(Boehringer Mannheim, Germany, catalog number 1732641) using thefollowing primers:

Primer ID JA53 (SEQ ID NO: 17)5′-GTGCGGCCGCACTCAAAGTTTATTGAGTTTACTTAGAG-3′; and Primer ID JA54 (SEQ IDNO: 18) 5′-ACAGATCTGTTTCTCACACTTGCAAAATTCTCTC-3′.

This third reaction is done to add the NotI and BglII restriction sitesto the 5′ and 3′ ends of the clone, respectively.

Following the reactions, a 25 μl aliquot of the High Fidelity PCRproduct is taken for agarose gel analysis. The band in the gel matchedthe expected size (˜1.3 kb) and is purified following the proceduredetailed in the Qiagen Gel Extraction Kit (Qiagen, Valencia, Calif.;product #28704). An aliquot of the purified PCR product is then digestedwith restriction enzymes NotI and BglII (Promega, Madison, Wis.) andligated into a pMON8677 backbone (FIG. 1), which has been cut with NotIand BglII to remove the e35S promoter cassette. The ligation is thenperformed with the Rapid DNA Ligation Kit from Boehringer-Mannheim(Germany, catalog number 1635379) according to the manufacturer'srecommendations. The resulting construct, named pMON42320 (FIG. 4), hadthe Sle2 promoter directly upstream of the uidA (β-glucuronidase)reporter gene.

Bacterial transformation is done as described in example 1. Purifiedplasmid containing the predicted insert size based on restriction enzymeanalysis is sequenced using the following primers:

JA51 (SEQ ID NO: 19) 5′-ATAGTACCCCAACACGCTAC-3′; JA53 (SEQ ID NO: 20)5′-GTGCGGCCGCACTCAAAGTTTATTGAGTTTACTTAGAG-3′; (SEQ ID NO: 18) JA54; JA55(SEQ ID NO: 21) 5′-GGAACCGAATATAATTGGCTC-3′; andthe GUS5′-2 primer (SEQ ID NO: 12), which anneals in the GUS codingregion of the plasmid.

Example 4

The Soybean lectin promoter is isolated from soybean genomic DNA (cv.3237) using PCR methodology. The PCR reaction is conducted using theExpand High Fidelity PCR System (Boehringer Mannheim, Germany, catalognumber Cat#1732641). The primary PCR reaction is performed using thefollowing primers, which are based on the published sequence (Vodkin et.al., Cell, 34:1023-1231 (1983)).

Primer ID JA23 5′-GCCTTCCTGGTCAGTAGCACCAGTA-3′; [SEQ ID NO: 22] andPrimer ID JA25 5′-CCATGCCATCGTATCGTGTCACAAT-3′. [SEQ ID NO: 23]

The oligonucleotide primers are supplied by Gibco Life Technologies(Grand Island, N.Y.). Following the primary PCR reaction, a nested PCRreaction is performed using 1 μl of product from the primary reactionand the following primers:

Primer ID JA26 (SEQ ID NO: 24)5′-AGGCGGCCGCCTGCAGATGGAATACAGCAATGAACAAATGC-3′; and Primer ID JA28 (SEQID NO: 25) 5′-CTCCATGGAGATCTTGCTTTGCTTCAGCTAAATTGCACT-3′.

This reaction is done to provide added cloning sites for the restrictionenzymes NotI and NcoI to the 5′ and 3′ ends of the cloned promoter,respectively.

The PCR components and conditions for the primary reaction are outlinedbelow:

Primary PCR

Component Amount Soybean Genomic DNA  2.0 μl dNTP mix (10 mM of eachdNTP)  1.0 μl Primer JA23 (10 μM)  1.5 μl Primer JA25 (10 μM)  1.5 μl10X PCR Buffer #3 (containing MgCl₂)   5 μl (final conc of 1X)Polymerase enzyme mix 0.75 μl Distilled Water bring to 50 μl finalvolume

The primary PCR reaction is initiated by denaturing the sample for 2minutes at 94° C. The reaction mixture is incubated for ten cyclesconsisting of 94° C. for 15 seconds, followed by 52° C. for 30 seconds,and finally 72° C. for 1 minute. The reaction is then incubated for 20cycles of 52° C. for 30 seconds, followed by 72° C. for 1 minute andincreasing the time by 20 seconds each cycle. The reaction is thenincubated at 72° C. for 7 minutes as a final extension and held at 4° C.for an extended incubation.

The PCR components and conditions for the nested PCR reaction areoutlined below:

Nested PCR

Component Amount Primary PCR product  1.0 μl dNTP mix (10 mM of eachdNTP)  1.0 μl Primer JA26 (10 μM)  1.5 μl Primer JA28 (10 μM)  1.5 μl10X PCR Buffer #3 (containing MgCl₂)   5 μl (final conc of 1X)Polymerase enzyme mix 0.75 μl Distilled Water bring to 50 μl finalvolume

The nested PCR reaction is initiated by denaturing the sample for 2minutes at 94° C. The reaction mixture is incubated for ten cyclesconsisting of 94° C. for 15 seconds, followed by 46° C. for 30 seconds,and finally 72° C. for 1 minute. The reaction is then incubated for 20cycles of 94° C. for 15 seconds, followed by 72° C. for 1 minute andincreasing the time by 20 seconds each cycle. The reaction is thenincubated at 72° C. for 7 minutes as a final extension and held at 4° C.for an extended incubation until the next step.

After nested PCR, the entire product of the High Fidelity PCR reactionis analyzed by agarose gel analysis. The 0.96Kb band is cut out andpurified using the Qiagen Gel Extraction Kit (Qiagen, Valencia, Calif.;product Cat#28704). An aliquot of the purified PCR product is thendigested with restriction enzymes NotI and NcoI (Promega, Madison, Wis.)and ligated into a pMON8677 backbone (FIG. 1), which is also cut withNotI and NcoI to remove the e35S promoter cassette (NBP# 6301633). Theligation is performed with the Rapid DNA Ligation Kit(Boehringer-Mannheim, Germany, catalog number Cat#1635379) according tothe manufacturer's recommendations. The resulting construct, in whichthe lectin promoter is directly upstream of the uidA (β-glucuronidase)reporter gene, is named pMON42302 (FIG. 5).

An aliquot of the ligation reaction is transformed into a suitable E.coli (DH5α) and the cells are plated on selection medium (LB with 100μg/ml ampicillin). Bacterial transformants are selected, grown in liquidculture (LB with 100 μg/ml ampicillin), and the plasmid DNA is isolatedusing the Qiaprep Spin Microprep Kit (Qiagen Corp., Valencia, Calif.).Purified plasmid containing the predicted insert size based onrestriction enzyme analysis are sequenced using the dye terminatormethod using primers: JA26, JA28, and GUS5′-2, which anneals in the GUScoding region of the plasmid. The sequence of the cloned lectin promotermatched that of the published lectin promoter from Vodkin, et al.,(1983).

Example 5

This example describes the transformation of soybean plants withheterologous genes driven by the sle2 and lectin promoters.

Vector Construction

Two Agrobacterium transformation vectors are constructed by followingstandard molecular cloning protocols (Sambrook et al., MolecularCloning—A laboratory manual, 1989, Cold Spring Harbor Laboratory Press;Maliga et al., Methods in Plant Molecular Biology—A laboratory coursemanual, 1995, Cold Spring Harbor Laboratory Press). An expressioncassette consisting of FMV:Elf1α promoter, CTP2 and CP4 coding gene andE9 3′UTR is included as selectable marker in two of the vectors,pMON66893 (FIG. 10), and pMON66894 (FIG. 11). The third vector pMON63682(FIG. 13) uses an expression cassette consisting of FMV promoter, CTP2and CP4 coding gene and E9 3′UTR for a selectable marker. In pMON66893(FIG. 10), the lectin promoter is ligated upstream of a gene consistingof chloroplast transit peptide CTP1 and an agrobacterium anthranilatesynthase (AgroAS). A NOS 3′ UTR is used to signal transcriptiontermination and polyadenylation. Vector pMON66894 (FIG. 11) isconstructed similarly to pMON66893, except that the sle2 promoter isused in place of the lectin promoter. Vector pMON63682 (FIG. 13) is alsoconstructed similarly to pMON66893, except that the Lea9 promoter isused in place of the lectin promoter.

Agrobacterium tumefaciens Mediated Transformation of Soybeans

The vectors described above are transferred into Agrobacteriumtumefaciens, strain ABI by a triparental mating method (Ditta et al.,Proc. Natl. Acad. Sci., 77:7347-7351 (1980)). The bacterial cells areprepared for transformation by methods well known in the art.

Commercially available soybean seeds (Asgrow A3244) are germinated overa 10-12 hour period. The meristem explants are excised and placed in awounding vessel and wounded by sonication. Following wounding, theAgrobacterium culture described above is added and the explants areincubated in for approximately one hour. Following inoculation, theAgrobacterium culture is removed by pipetting and the explants areplaced in co-culture for 2-4 days. The explants are then transferred toselection media consisting of Woody Plant Medium (McCown and Lloyd,Proc. International Plant Propagation Soc., 30:421, (1981)), plus 75 μMglyphosate and antibiotics to control Agro overgrowth) for 5-7 weeks toallow selection and growth of transgenic shoots. Phenotype positiveshoots are harvested approximately 5-7 weeks post inoculation and placedinto selective rooting media comprising Bean Rooting Media (BRM) with 25μM glyphosate (Martinell, et al., U.S. Pat. No. 5,914,451) for 2-3weeks. Shoots producing roots are transferred to the greenhouse andpotted in soil. Shoots that remain healthy on selection, but do notproduce roots are transferred to non-selective rooting media (i.e., BRMwithout glyphosate) for an additional two weeks. Roots from any shootsthat produce roots off selection are tested for expression of the plantselectable marker before they are transferred to the greenhouse andpotted in soil. Plants are maintained under standard greenhouseconditions until R1 seed harvest.

Free Amino Acid Analysis

The levels of free amino acids are analyzed from each of the transgenicevents using the following procedure. Seeds from each of the transgenicevents are crushed individually into a fine powder and approximately 50mg of the resulting powder is transferred to a pre-weighed centrifugetube. The exact sample weight of the sample is recorded and 1.0 ml of 5%trichloroacetic acid is added to each sample tube. The samples are mixedat room temperature by vortex and then centrifuged for 15 minutes at14,000 rpm on an Eppendorf microcentrifuge (Model 5415C, BrinkmannInstrument, Westbury, N.Y.). An aliquot of the supernatant is removedand analyzed by HPLC (Agilent 1100) using the procedure set forth inAgilent Technical Publication “Amino Acid Analysis Using the ZorbaxEclipse-AAA Columns and the Agilent 1100 HPLC,” Mar. 17, 2000.

The results indicate that transformed soybeans, which have the AgroASgene driven by the lea9, Lectin and the sle2 promoters, have higherlevels of free tryptophan in their seeds.

Example 6

This example describes the analysis of gene expression under the controlof the lea9 and per1 promoters in transformed soybean plants, to testthe effectiveness of the lea9 and per1 promoters in driving heterologousgenes in transgenic soybean plants resulting in elevated levels of aminoacids in the seeds.

Vector Construction

Four Agrobacterium transformation vectors are constructed by followingstandard molecular cloning protocols (Sambrook et al., MolecularCloning—A laboratory manual, 1989, Cold Spring Harbor Laboratory Press;Maliga et al., Methods in Plant Molecular Biology—A laboratory coursemanual, 1995, Cold Spring Harbor Laboratory Press). An expressioncassette consisting of FMV promoter (with HSP70 Leader sequence), CTP2and CP4 coding gene and E9 3′ UTR is included as selectable marker inall four vectors. In pMON69650 (FIG. 6), the lea9 promoter is ligatedupstream of a tryptophan-insensitive α-subunit of anthranilate synthasefrom C28 maize (Anderson et al., U.S. Pat. No. 6,118,047). A NOS 3′ UTRis used to signal transcription termination and polyadenylation. VectorpMON69651 (FIG. 7) is constructed similarly to pMON69650, except thatthe per1 promoter is used in place of lea9, driving the sametryptophan-insensitive α-subunit of anthranilate synthase from C28maize. Vector pMON63662 (FIG. 12) contains the same genetic elements aspMON69651, except that two sets of borders are used to separate the geneof interest and the selectable marker, allowing for independentsegregation of the marker and anthranilate synthase genes.

The vectors pMON64210 (FIG. 8) and pMON64213 (FIG. 9) are designed todemonstrate simultaneous deregulation of multiple pathways. BothpMON64210 and pMON64213 contain a 7Sα′ promoter driving thetryptophan-insensitive a-subunit of anthranilate synthase from C28maize, with the NOS 3′ UTR. Additionally, pMON64210 contains anexpression cassette consisting of a lea9 promoter driving a fusionprotein of Arabidopsis SSU CTP and an Ile-insensitive threoninedeaminase (Gruys et al., U.S. Pat. No. 5,942,660) with Arc5 3′ UTR. Thevector pMON64213 is constructed similarly to pMON64210 except per1promoter is used in place of lea9, driving the sametryptophan-insensitive α-subunit of anthranilate synthase from C28maize.

Agrobacterium tumefaciens Mediated Transformation of Soybeans

The vectors described above are transferred into Agrobacteriumtumefaciens, strain ABI by a triparental mating method (Ditta et al.,Proc. Natl. Acad. Sci., 77:7347-7351 (1980)). The bacterial cells areprepared for transformation by methods well known in the art.

Commercially available soybean seeds (Asgrow A3244) are germinated overa 10-12 hour period. The meristem explants are excised and placed in awounding vessel and wounded by sonication. Following wounding, theAgrobacterium culture described above is added and the explants areincubated in for approximately one hour. Following inoculation, theAgrobacterium culture is removed by pipetting and the explants areplaced in co-culture for 2-4 days. The explants are then transferred toselection media consisting of Woody Plant Medium (McCown and Lloyd,Proc. International Plant Propagation Soc., 30:421, (1981)), plus 75 μMglyphosate and antibiotics to control Agro overgrowth) for 5-7 weeks toallow selection and growth of transgenic shoots. Phenotype positiveshoots are harvested approximately 5-7 weeks post inoculation and placedinto selective rooting media comprising Bean Rooting Media (BRM) with 25μM glyphosate (Martinell, et al., U.S. Pat. No. 5,914,451) for 2-3weeks. Shoots producing roots are transferred to the greenhouse andpotted in soil. Shoots that remain healthy on selection, but do notproduce roots are transferred to non-selective rooting media (i.e., BRMwithout glyphosate) for an additional 2 weeks. Roots from any shootsthat produce roots off selection are tested for expression of the plantselectable marker before they are transferred to the greenhouse andpotted in soil. Plants are maintained under standard greenhouseconditions until R1 seed harvest.

Free Amino Acid Analysis

The levels of free amino acids are analyzed from each of the transgenicevents using the following procedure. Seeds from each of the transgenicevents are crushed individually into a fine powder and approximately 50mg of the resulting powder is transferred to a pre-weighed centrifugetube. The exact sample weight of the sample is recorded and 1.0 ml of 5%trichloroacetic acid is added to each sample tube. The samples are mixedat room temperature by vortex and then centrifuged for 15 minutes at14,000 rpm on an Eppendorf microcentrifuge (Model 5415C, BrinkmannInstrument, Westbury, N.Y.). An aliquot of the supernatant is removedand analyzed by HPLC (Agilent 1100) using the procedure set forth inAgilent Technical Publication “Amino Acid Analysis Using the ZorbaxEclipse-AAA Columns and the Agilent 1100 HPLC,” Mar. 17, 2000.

The data from this analysis is shown in the tables below. Because the R1seeds from each event represent a population of segregating seeds, theseed with the highest tryptophan level among the 10 seeds analyzed perevent is chosen as a representative of a homozygous genotype. Tenrandomly selected non-transgenic seeds from Asgrow A3244 are alsoanalyzed. The seed with the highest tryptophan level is chosen as thenegative control. The data indicate that an increase in tryptophanlevels is observed when the lea9 and per1 promoters are driving theexpression of the maize ASα gene (pMON69650 and pMON69651,respectively). Both tryptophan and isoleucine levels are increased intransformed plants having the lea9 and per1 promoters driving the TDgene, (pMON64210 and pMON64213, respectively).

Tryptophan Accumulation in Transgenic Soybean Seeds Using pMON69650

Event No. Trp (ppm) A3244 307 pMON69650- 1 3548 2 2339 3 990 4 2983 52455 6 2761 7 2720 8 3949 9 1865 10 407 11 2880 12 2062 13 3170 14 350415 1723 16 1260 17 708 18 1878 19 1490 20 2437 21 7160 22 2792 23 129224 2333 25 696 26 1513 27 2390 28 3636 29 871 30 433 31 2122 32 2116 33665 34 435

Tryptophan Accumulation in Transgenic Soybean Seeds Using pMON69651

Event No. Trp (ppm) A3244 307 pMON69651- 1 3249 2 3284 3 4649 4 2808 53832 6 2264 7 4400 8 2854 9 5160 10 5479 11 2647 12 4079 13 2994 14 394615 5511 16 515 17 958 18 2764

Tryptophan and Isoleucine Accumulation in Transgenic Soybean Seeds UsingpMON64210

Event No. ILE (ppm) TRP (ppm) A3244 182.4 86.9 pMON64210-  1 8829.6774.7  2 14933.3 1763.3  3 21474.8 1928.1  4 21892.9 2500.0  5 21117.82489.4  6 20639.7 2451.2  7 1843.1 347.8  8 19124.1 1755.5  9 10000.01395.5 10 15728.8 1305.1 11 21992.6 1988.9 12 17530.9 2034.0

Tryptophan and Isoleucine Accumulation in Transgenic Soybean Seeds UsingpMON64213

Event No. ILE (ppm) TRP (ppm) A3244 182.4 86.9 pMON64210-  1 8829.6774.7  2 14933.3 1763.3  3 21474.8 1928.1  4 21892.9 2500.0  5 21117.82489.4  6 20639.7 2451.2  7 1843.1 347.8  8 19124.1 1755.5  9 10000.01395.5 10 15728.8 1305.1 11 21992.6 1988.9 12 17530.9 2034.0

Trp Levels in Three Marker Free Events Carrying Per1-Maize AS frompMON63662

Construct Event Line No Average/event MaxTrp pMON63662 GM_A29574 28 10761723 pMON63662 GM_A29578 44 2129 3837 pMON63662 GM_A29672 76 1188 1549

Trp Levels in Three Marker Free (Two Positive and a Null) Lines CarryingLea9-wt-Agro AS from pMON63682

Avg (trp > Construct Pedigree Event 450) MaxTrp pMON63682GM_A30383:@.0030. GM_A30383 1678 2,018 pMON63682 GM_A30383:@.0043.GM_A30383 2064 2,820 pMON63682 GM_A30383:@.0084. GM_A30383 0 46References

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1. An isolated nucleic acid molecule comprising SEQ ID NO: 1 or afragment thereof with promoter activity, or a full complement of SEQ IDNO:1 or said fragment thereof.
 2. A nucleic acid construct comprising apromoter operably linked to a second nucleic acid, wherein the promotercomprises SEQ ID NO: 1, or a fragment thereof with promoter activity, ora full complement of SEQ ID NO:1 or said fragment thereof.
 3. A plantcomprising the nucleic acid construct of claim
 2. 4. The plant of claim3, wherein said second nucleic acid is a structural nucleic acid.
 5. Theplant of claim 4, wherein said structural nucleic acid encodes a proteinselected from the group consisting of a seed storage protein, a fattyacid pathway enzyme, a tocopherol biosynthetic enzyme, an amino acidbiosynthetic enzyme, a steroid pathway enzyme, and a starch branchingenzyme.
 6. The plant of claim 4, wherein said structural nucleic acidencodes a protein selected from the group consisting of anthranilatesynthase, tryptophan decarboxylase, threonine deaminase, and aspartatekinase.
 7. The plant of claim 4, wherein said structural nucleic acidencodes a starch branching enzyme.
 8. The plant of claim 4, wherein saidstructural nucleic acid is oriented to express an antisense RNAmolecule.
 9. The plant of claim 3, wherein said plant is selected fromthe group consisting of canola, crambe, mustard, castor bean, sesame,cottonseed, linseed, maize, soybean, Arabidopsis Phaseolus, peanut,alfalfa, wheat, rice, oat, sorghum, rapeseed, rye, tritordeum, millet,fescue, perennial ryegrass, sugarcane, cranberry, papaya, banana,safflower, oil palms, flax, muskmelon, apple, cucumber, dendrobium,gladiolus, chrysanthemum, liliacea, cotton, eucalyptus, sunflower,Brassica campestris, Brassica napus, turfgrass, sugarbeet, coffee, anddioscorea.
 10. The plant of claim 3, wherein said plant is soybean. 11.The plant of claim 4, wherein said structural nucleic acid is expressedin an organ specific manner.
 12. The plant of claim 11, wherein saidstructural nucleic acid is expressed in a seed.
 13. A method ofproducing a transformed plant comprising: (a) providing the nucleic acidconstruct of claim 2, wherein said second nucleic acid is a structuralnucleic acid; and (b) transforming a plant with said nucleic acidconstruct.
 14. The method of claim 13, wherein said plant produces aseed and said structural nucleic acid is transcribed in said seed.
 15. Ameal comprising the plant of claim
 12. 16. A feedstock comprising theplant of claim
 12. 17. A cell containing the nucleic acid construct ofclaim
 2. 18. The cell according to claim 17, wherein said cell isselected from the group consisting of a bacterial cell, a mammaliancell, an insect cell, a plant cell, and a fungal cell.
 19. The cellaccording to claim 18, wherein said cell is Agrobacterium tumefaciens.20. A seed generated by a plant containing the nucleic acid construct ofclaim
 2. 21. A method for expressing a structural nucleic acid in aplant, comprising: (a) expressing in said plant a nucleic acid moleculecomprising a promoter operably linked to said structural nucleic acid,wherein said promoter comprises a polynucleotide comprising SEQ ID NO:1, or a fragment thereof with promoter activity, or a full complement ofSEQ ID NO:1 or said fragment thereof, and wherein said promoter and saidstructural nucleic acid are heterologous with respect to each other; and(b) cultivating said plant.
 22. The method of claim 21, wherein saidstructural nucleic acid is in the anti-sense orientation.
 23. A methodof accumulating free amino acids in a seed of a plant, comprising: (a)transforming said plant with the nucleic acid construct of claim 2,wherein the second nucleic acid encodes an amino acid biosynthesis gene,and wherein said promoter and said second nucleic acid are heterologouswith respect to each other; and (b) growing said plant.
 24. A method ofcontrolling the germination of a soybean seed, comprising: (a)transforming a plant with the nucleic acid construct of claim 2, whereinthe second nucleic acid encodes a gibberellin biosynthetic polypeptide,and wherein said promoter and said second nucleic acid are heterologouswith respect to each other; (b) growing said plant; (c) harvesting seedfrom said plant; and (d) planting said seed.